Method for Producing Carboxylic Acid Using Methanol-Assimilating Bacterium

ABSTRACT

A method for producing a carboxylic acid by a fermentation process which comprises culturing a methanol-assimilating bacterium capable of producing the carboxylic acid in a liquid medium containing methanol and a counter ion to produce and accumulate the carboxylic acid in the medium, further comprising the feeding of a substance comprising methanol and a counter ion to the medium by fed-batch culturing to maintain the total ionic strength within the fermentation medium at or below a certain level.

This application is a Divisional of, and claims priority under 35 U.S.C.§120 to, U.S. patent application Ser. No. 12/241,293, filed Sep. 30,2008, which was a continuation of, and claimed priority under 35 U.S.C.§120 to, PCT Patent Application No. PCT/JP2007/057524, filed Mar. 28,2007, which claims priority under 35 U.S.C. §119 to Japanese PatentApplication No. 2006-095207, filed Mar. 30, 2006, all of which areincorporated by reference herein. The Sequence Listing filedelectronically herewith is also hereby incorporated by reference in itsentirety (File Name: 2013-06-17T_US-375D_Seq_List; File Size: 74 KB;Date Created: Jun. 17, 2013).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the microbiology industries, and moreparticularly, to a method of producing carboxylic acid by fermentation.The present invention also relates to the microorganism which is used inthe production method

2. Brief Description of the Related Art

L-amino acids such as L-lysine, L-glutamic acid, L-threonine, L-leucine,L-isoleucine, L-valine, and L-phenylalanine are industrially produced byfermentation using microorganisms belonging to the genus Brevibacterium,Corynebacterium, Bacillus, Escherichia, Streptomyces, Pseudomonas,Arthrobacter, Serratia, Penicillium, Candida, and the like. To enhanceproductivity, strains of microorganisms are employed which have beenisolated from nature and artificial variants thereof. Moreover, varioustechnologies have been disclosed which increase the production ofL-amino acids, such as recombinant DNA technology.

Methanol is an economical and commercially available raw material usedin the conventional fermentation methods of carboxylic acid.Microorganisms employed in such methods include those belonging to thegenera Achromobacter, Pseudomonas (Japanese Patent ApplicationPublication No. Showa 45-25273), Protaminobacter (Japanese PatentPublication No. Showa 49-125590), Protaminobacter, Methanomonas(Japanese Patent Application Publication No. Showa 50-25790),Microcyclus (Japanese Patent Application Publication No. Showa52-18886), Methylobacillus (Japanese Patent Application Publication No.Heisei 4-91793), and Bacillus (Japanese Patent Application PublicationNo. Heisei 3-505284).

Thus far, the use of artificial mutations or recombinant DNA techniqueshave been developed and used in a method of producing L-amino acidsusing Methylophilus bacteria. Specifically, the enzymatic activities ofdihydrodipicolinate synthase and/or aspartokinase were increased(International Publication No. 00/61723). The amino acid export processhas been a major obstacle in the production of amino acids byfermentation from methanol using methanol-assimilating bacteria. Toovercome this problem, a mutant of the LysE protein, which participatesin the export of L-lysine, exhibits L-lysine export activity inmethanol-assimilating bacteria, and was isolated from bacteria of thegenus Corynebacterium. Therefore, by breeding using artificialmutations, recombinant DNA techniques, and this modified L-lysine exportcarrier, it is possible to efficiently produce L-lysine. Hence, a methodfor producing L-amino acid using bacteria of the genus Methylophilus wasdeveloped (International Publication No. 00/61723 and Japanese PatentPublication No. 2004-166594).

Through the breeding of microorganisms such as discussed above, theability to produce substances using methanol-assimilating bacteria hasbeen greatly improved. However, there is still a need for thedevelopment of inexpensive and efficient methods of producing carboxylicacids from methanol.

SUMMARY OF THE INVENTION

The present invention provides a method for producing carboxylic acids,particularly L-amino acids, with high efficiency from methanol, which isinexpensive and abundantly available.

It was noticed that during fermentation using methanol-assimilatingbacteria and methanol as the carbon source, a high ionic strengthinhibited the proliferation of the bacteria. By feeding the culturewhile keeping the rate of increase in ionic strength in the culturemedium at or below a certain level, a high production rate could beachieved without inhibiting the growth of the bacteria.

It is an aspect of the present invention to provide a method forproducing carboxylic acid by fermentation comprising culturing amethanol-assimilating bacterium that has an ability to producecarboxylic acid in a liquid fermentation medium containing methanol anda counter ion, and collecting the carboxylic acid from the medium or thebacterium, wherein the total ionic strength is controlled to be apredetermined level or less in the fermentation medium by feeding acomposition comprising methanol and a counter ion to the medium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the rate of increase of the total ionicstrength in the medium is 0.02 mol/m³/hour or less.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the ionic strength is controlled during theproliferation period of the methanol-assimilating bacterium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said substance is selected from the groupconsisting of ammonium sulfate, ammonium chloride, ammonium glutamate,ammonium succinate, ammonium fumarate, ammonium aspartate, urea, andcombinations thereof.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said carboxylic acid is an L-amino acid.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said L-amino acid is L-lysine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said methanol-assimilating bacterium is belongsto a genera selected from the group consisting of the genusAcromobacter, Pseudomonas, Protaminobacter, Methanomonas, Microcyclus,Methylobacillus, Bacillus, and Methylophilus.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said methanol-assimilating bacterium has beenmodified to increase the activity of an enzyme selected from the groupconsisting of diaminopimelate dehydrogenase, diaminopimelatedecarboxylase, aspartate semialdehyde dehydrogenase, and combinationsthereof.

It is a further aspect of the present invention to provide the method asdescribed above, wherein DNA encoding dihydrodipicolinate synthaseand/or aspartokinase that has been modified so it is not subject tofeedback inhibition by L-lysine is present in said methanol-assimilatingbacterium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein DNA encoding mutant lysE protein which promotesthe export of L-lysine to the outside of the bacterium is present insaid methanol-assimilating bacterium.

It is a further aspect of the present invention to provide a method forproducing carboxylic acid by fermentation comprising culturing amethanol-assimilating bacterium that has an ability to produce thecarboxylic acid in a liquid medium containing methanol and a counterion, anc collecting the carboxylic acid from the medium or thebacterium, wherein a composition comprising methanol and a counter ionis fed to the medium by fed-batch culturing.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said counter ion is a monovalent ion.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said counter ion is selected from the groupconsisting of ammonium chloride, ammonium glutamate, ammonium succinate,ammonium fumarate, ammonium aspartate, and combinations thereof.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said carboxylic acid is an L-amino acid.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said L-amino acid is L-lysine.

It is a further aspect of the present invention to provide the method asdescribed above), wherein said methanol-assimilating bacterium is of agenera selected from the group consisting of Acromobacter, Pseudomonas,Protaminobacter, Methanomonas, Microcyclus, Methylobacillus, Bacillus,and Methylophilus.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said methanol-assimilating bacterium has beenmodified to increase the activity of an enzyme selected from the groupconsisting of diaminopimelate dehydrogenase, diaminopimelatedecarboxylase, aspartate semialdehyde dehydrogenase, and combinationsthereof.

It is a further aspect of the present invention to provide the method asdescribed above, wherein DNA encoding dihydrodipicolinate synthaseand/or aspartokinase that has been modified so it is not subject tofeedback inhibition by L-lysine is present in the bacterium.

It is a further aspect of the present invention to provide the method asdescribed above, DNA encoding a variant lysE protein promoting theexport of L-lysine to the outside of the cell when introduced into amethanol-assimilating bacterium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of the plasmid pM12.

FIG. 2 shows the construction of the pMIV5.

FIG. 3 shows the construction of the plasmid pMIV-FRTmFRT.

FIG. 4 shows the construction of the plasmid pAET7.

FIG. 5 shows the construction of the plasmid pFLP31.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

<1> Production Method

A method of producing a carboxylic acid by culturing amethanol-assimilating bacterium having the ability to produce acarboxylic acid in a liquid medium containing methanol and a counter ionis described. More specifically, during the culture, methanol and thecounter ion are added to the medium by a fed-batch method to control thetotal ionic strength at a predetermined level. The term “total ionicstrength” means the strength of all ions which are present in themedium. The phrase “all ions contained in the medium” means all thecations and anions in the medium which contain carboxylic acid,carboxylic acid counter ions, other organic acids, and the like.

The term “carboxylic acid” means a substance having a carbon structurein the form of a carboxyl group (—CO₂H), including, for example, organicacids, fatty acids, amino acids, hydroxy acids, and keto acids. Thecarboxylic acid may be an L-amino acid, with no particular limitation onthe type of L-amino acid. Examples include basic amino acids such asL-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline;aliphatic amino acids such as L-isoleucine, L-alanine, L-valine,L-leucine, and L-glycine; amino acids in the form ofhydroxymonoaminocarboxylic acids, such as L-threonine and L-serine;cyclic amino acids such as L-proline; aromatic amino acids such asL-phenylalanine, L-tyrosine, and L-tryptophan; sulfur-containing aminoacids such as L-cysteine, L-cystine, and L-methionine; and acidic aminoacids such as L-glutamic acid, L-aspartic acid, L-glutamine, andL-asparagine. Of these, the basic L-amine acids L-lysine, L-asparagine,L-ornithine, and L-histidine are desirable.

The medium contains methanol as the carbon source, and is adjusted tocontain counter ions of carboxylic acid.

The composition or substance serving as the source of the counter ionsmay be capable of maintaining a low total ionic strength in the medium.A composition containing monovalent or divalent anions is desirable.Examples are ammonium sulfate, ammonium chloride, ammonium glutamate,ammonium succinate, ammonium fumarate, ammonium aspartate, and urea.

The composition containing methanol and a counter ion is desirably fedto the culture medium in a feed medium. However, the compositioncontaining methanol and counter ions may also be present in the initialmedium. The term “initial medium” indicates the medium which is used inthe batch culture prior to feeding any other medium, or the medium thatis fed to the culture. The term “feed medium” indicates the medium thatis fed into a fermentation vessel over the course of the fed-batchculture. The term “fermentation medium” indicates the medium used in thefermentation vessel, and the carboxylic acid is collected from thisfermentation medium. “Fermentation vessel” means the apparatus in whichthe carboxylic acid fermentation is conducted, and a fermentation tankor jar fermentor may be employed. The capacity of the vessel need onlybe sufficient for the production and collection of the carboxylic acid.

The ionic strength of the counter ions in the fermentation medium may becontrolled over the course of the culture, with the rate of increase inionic strength of the total counter ions in the medium maintained at orbelow a predetermined level. The feeding of the composition containingthe counter ions may be conducted while controlling the rate of rise inionic strength to 0.02 mol/m²/hour or less, desirably 0.015 mol/m²/houror less, and preferably, 0.01 mol/m²/hour or less.

The ionic strength may be limited to or less than a predetermined levelover the entire course of the culture, but may be so limited only duringcertain steps. For example, when there are periods of proliferation ofthe microorganism (proliferation period) and periods of production ofthe carboxylic acid (production period), the ionic concentration may belimited to or less than a predetermined concentration during theproliferation period. The ionic strength need not be kept within theabove-stated range at all times during the culture; it is possible thatthe amount of counter ions which are present at a certain period mayexceed the above-stated range, and then be reduced during culturing. Thecomposition containing counter ions can also be intermittently addedwhen the amount of counter ions decreases in fermentation. The term“proliferation period” indicates the period at the start of the culturewhen the carbon source is primarily used for bacterial growth. Thisperiod maybe about 10 hours, desirably 18 hours, and preferably 24hours, and is the period during which the microorganism undergoeslogarithmic growth. The “production period” indicates the periodbeginning 24 hours after the start of the culture during whichproduction of carboxylic acid occurs.

It is sufficient that the feed medium contain a minimum number ofcounter ions of carboxylic acid; however, sulfer may still temporarilyrun short. The term “temporarily,” for example, indicates the counterions may run short for a period of about 20 percent, 40 percent, or amaximum of 60 percent of the total duration of fermentation. During theperiod when the counter ions run short, and may temporarily be 0, therate of the increase in ionic strength may be 0.001 mol/m²/hour orhigher.

The rate of the increase in ionic strength in the fermentation mediumcan be calculated by measuring the quantity of sulfate ions, chlorideions, carbonate ions, and carboxylic acid. For example, the sulfate andchloride ions can be measured by ion chromatography, and the carbonateions can be measured with a carbonic acid gas measuring apparatus.

Other carbon sources may be present in the medium in addition tomethanol, such as glucose, glycerol, fructose, sucrose, maltose,mannose, galactose, starch hydrolysis products, molasses, and othersugars. Additionally, acetic acid, citric acid, and other organic acidsmay be present. Other raw materials which may be present and serve asthe carbon source include cane molasses, beet molasses, high testmolasses, and citrus molasses, as well as the hydrolysis products ofnatural raw materials such as cellulose, starch, corn, cereals, andtapioca. Carbon dioxide dissolved in the culture liquid can alsofunction as the carbon source. These carbon sources can be present inthe initial medium as well as in the feed medium. They may be mixed withmethanol in both the initial medium and feed medium, or the culture maybe conducted using only methanol as the carbon source in the feedmedium, and some other carbon source initially.

Methanol is desirably fed to the fermentation medium so that theproportion in the fermentation medium is 2 percent or less, desirably0.5 percent or less, and preferably, 0.2 percent or less.

The source of nitrogen in the medium may include counter ions, ammoniumsulfate, ammonium chloride, ammonium glutamate, other ammonium salts,nitrates, and the like. Ammonia gas and ammonia water which are used toadjust the pH are also sources of nitrogen. Peptones, yeast extracts,meat extracts, wheat germ extracts, corn steep liquor, soybeanhydrolysis products, and the like may also be present. These nitrogensources may be present in both the initial medium and feed medium. Thesenitrogen sources may be blended into both the initial medium and thefeed medium, or the nitrogen source present in the feed medium may bedifferent from that present in the initial medium.

In addition to a carbon source, nitrogen source, and sulfur source, aphosphorus source is also desirably present in the medium. Phosphorussources that are suitable for use include potassium dihydrogenphosphate, dipotassium hydrogen phosphate, pyrrolinic acid, and otherphosphoric acid polymers.

In addition to a carbon source, nitrogen source, and sulfur source, themedium may also contain a growth-promoting factor, such as trace metals,amino acids, vitamins, fatty acids, nucleic acids, peptones containingthe same, casamino acids, yeast extracts, and soy protein degradationproducts. Examples of trace metals include iron, manganese, magnesium,and calcium. Examples of vitamins include vitamin B1, vitamin B2,vitamin B6, nicotinic acid, nicotinamide, and vitamin B 12. Thesegrowth-promoting factors may be incorporated into the initial medium, ormay be incorporated into the feed medium.

Furthermore, when an auxotrophic mutant requires nutrients such as aminoacids or the like for growth, the required nutrients may be added to themedium. The addition of L-methionine and L-threonine when using L-lysineproducing bacteria is desirable because L-lysine producing bacteriaoften auxotrophic for L-methionine or L-threonine and/or contain adeletion which results in degradation of L-threonine (U.S. PatentApplication Publication 2004-0214296).

The culture may be conducted at a fermentation temperature of 20 to 45°C., preferably 33 to 42° C., with aeration. Here, the oxygenconcentration is adjusted to between 5 and 50 percent, desirably about10 percent. The pH is controlled to 5 to 9 and the culture is conductedwith aeration. When the pH is reduced during the culture, for example,calcium carbonate or an alkali such as ammonia gas or aqueous ammoniamay be added to neutralize the culture. Culturing under such conditions,desirably for about 10 to 120 hours, results in the accumulation of alarge amount of carboxylic acid in the culture solution. Theconcentration of the carboxylic acid is higher than in wild-typestrains; the concentration of L-lysine is not limited as long asL-lysine can be isolated and collected from the medium. Theconcentration is 10 g/L or greater, desirably 30 g/L or greater, andpreferably, 50 g/L or greater.

The carboxylic acid can be collected from the culture medium at the endof the culture by any known collection method. For example, afterremoving the bacterial mass from the culture solution by centrifugalseparation or the like, concentration precipitation can be used forcollection. L-lysine can be collected by combining ordinary ion-exchangeresin methods, precipitation methods, and other known methods.

The culture method may include batch culture, fed-batch culture, andcontinuous culture. To maintain the carboxylic acid accumulation at orabove a prescribed level in the present invention, the seed culture andmain culture can be conducted separately. The seed culture can beconducted as a shaking culture in a flask or the like, or as a batchculture. The main culture can be conducted as fed-batch culture orcontinuous culture. Both the seed culture and main culturing can beconducted as batch culture.

During the course of a fed-batch culture or continuous culture, thefeeding of methanol and nutrient sources may be temporarily suspended.During the period of maximum feeding, feeding is stopped for 30 percentof the time or less, desirably 20 percent of the time or less, andpreferably, 10 percent of the time or less. When the feed medium isintermittently added, the feed medium may be initially added over apredetermined level, and the second and following additions may occurwhen an increase in the pH or the dissolved oxygen concentration isdetected by a computer (U.S. Pat. No. 5,912,113).

A method of producing a carboxylic acid is described by fermentation ofa methanol-assimilating bacterium having the ability to produce acarboxylic acid in a liquid medium containing methanol and counter ions.By this method, carboxylic acid is produced in the same medium, and thecounter ion is a monovalent ion, such as ammonium chloride, ammoniumglutamate, ammonium succinate, ammonium fumarate, and ammoniumaspartate.

<2> The Methanol-Assimilating Bacteria

The term “methanol-assimilating bacterium” indicates a bacterium that iscapable of growing using methanol as its main carbon source. Specificexamples include bacteria of the genus Methylophilus, such asMethylophilus methylotrophus, bacteria of the genus Methylobacillus,such as Methylobacillus glycogenes and Methylobacillus flagellatus,bacteria of the genera Acromobacter and Pseudomonas (JP45-25273A),bacteria of the genus Protaminobacter (JP49-125590A), bacteria of thegenera Protaminobacter and Methanomonas (JP50-25790A), and bacteria ofthe genus Microcyclus (JP52-18886A). Bacteria of the genusMethylobacterium may also be employed.

Examples of Methylophilus methylotrophus include the AS 1 (NCIMB 10515)and W3A1 (NCIMB 11348) strains. Methylophilus methylotrophus AS1(NCIMB10515) and W3A1 (NCIMB 11348) are available from the NationalCollections of Industrial and Marine Bacteria, NCIMB Lts., Tony ResearchStation 135, Abbey Road, Aberdeen AB9 8DG, United Kingdom.

Examples of Methylobacillus glycogenes include the T-11 (NCIMP 11375),ATCC 21276, ATCC 21371, ATR80 (Appl. Microbiol. Biotechnol., (1994),Vol. 42, pp. 67-72), and A513 (Appl. Microbiol. Biotechnol., (1994),Vol. 42, pp. 67-72) strains. The Methylobacillus glycogenes NCIMB 11375strain is available from the National Collections of Industrial andMarine Bacteria, NCIMB Lts., Torry Research Station 135, Abbey Road,Aberdeen AB9 8DG, United Kingdom.

Examples of Methylobacillus flagellatus include the ATTC 51484, KT (N.I. Govorukhina et al., Microbiology (Russia) 56 (1987), pp. 849-854),and VKM B-1610 strains. Methylobacillus flagellatus VKM B-1610 isavailable from the All-Russian Collection of Microorganisms (Russia,142290, Moscow Region, Pushchino, pr. Nauki, 5, IBPM).

The Methylobacillus glycogenes ATCC 21276 and ATCC 21371, and theMethylobacillus flagellatus ATTC 51484 strains may be obtained from theAmerican Type Culture Collection (ATCC) (ATCC, Address: P.O. Box 1549,Manassas, Va. 20108, 1, USA).

The term “capable of producing a carboxylic acid” means the ability toproduce and secrete free carboxylic acid into the medium, that is,outside the cell, when cultured in the medium, and particularly meansthe ability to produce more carboxylic acid than the wild-type strain(parent strain). Examples of the wild-type strain include the AS 1strain (NCIMB 10515) for bacteria of the genus Methylophilus and theT-11 strain (NCIMB 11375) for bacteria of the genus Methylobacillus.

To obtain the ability to produce a carboxylic acid, a conventionalmethod for breeding Escherichia bacteria may be used, such as byacquiring an auxotrophic mutant, analog resistant, strain, or metaboliccontrol mutant strain, as well as by constructing a recombinant strainwith enhanced activity of a carboxylic acid biosynthesis enzyme. (seeAmino Acid Fermentation, Gakkai Shuppan Center, 1st ed. May 30, 1986,pp. 77-100). Therefore, when breeding carboxylic acid-producingbacteria, one or more properties such as an auxotrophic mutant, analogresistant, or metabolically controlled mutation may be imparted. Theexpression of one or more carboxylic acid biosynthesis-related enzymesmay be increased. The acquisition of an auxotrophic mutant, analogresistance, or metabolically controlled mutation may be combined withthe strengthening of a carboxylic acid biosynthesis-related enzyme.

Strains with an auxotrophic mutant, carboxylic acid analog-resistantstrains, and variants with controlled metabolisms that are capable ofproducing carboxylic acid can be obtained by subjecting a parent strainor wild-type strain to a known mutagenic treatment, such as byirradiation with X-rays or ultraviolet radiation, or by treatment with amutagenic agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) orethyl methane sulfonate (EMS), and selecting those strains that exhibitboth a auxotrophic mutant, analog resistance, or metabolicallycontrolled mutation and the ability to produce a carboxylic acid fromamong the variants.

Examples of gene recombination include increasing the expression of thegene encoding an enzyme involved in the biosynthesis of the targetcarboxylic acid and reducing the activity of the gene encoding an enzymeinvolved in degradation of the targeted carboxylic acid.

Specific examples of microorganisms which are able produce a carboxylicacid, and particularly microorganisms which are also able to produceL-amino acids, are described below. However, the microorganisms that canbe employed are not limited to these.

<2-1> Imparting the Ability to Produce L-Lysine

Methanol-assimilating bacteria which are able to produce L-lysine, suchas strains of Methylophilus methylotrophus, can be obtained bysubjecting a strain which is not able to produce, or produces verylittle, L-lysine to a mutagenic treatment to impart resistance to alysine analog such as S-(2-aminoethyl)-L-cysteine (“AEC” hereinafter).Methods of mutagenic treatment include subjecting the bacterial strainto a physical stimulus such as UV radiation, X-ray, or γ-rays, ortreating it with a chemical mutagenic agent such as NTG. Methylophilusmethylotrophus AJ13608 is an example of a bacterial strain of the genusMethylophilus which is able to produce L-lysine that was obtained inthis manner.

The original bacterial strain was bred by imparting AEC-resistance tothe Methylophilus methylotrophus AS1 strain. Methylophilusmethylotrophus AJ13608 was deposited as deposit number FERM P-17416 withthe National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology (now the International Patent OrganismDepositary (Chuo No. 6, 1-banchi, 1-chome, Tsukuba-shi Higashi,Ibaraki-ken, Japan, Postal Code 305-8566) of the National Institute ofAdvanced Industrial Science and Technology, an IndependentAdministrative Institution) on Jun. 10, 1999, and was converted tointernational deposit under the Budapest Treaty on Mar. 31, 2000, withthe designation FERM BP-7112.

Methanol-assimilating bacteria which are able to produce L-lysine canalso be bred using a gene recombination technique to introduce andincrease DNA encoding genes involved in the biosynthesis of L-lysine.These genes include those encoding enzymes of the L-lysine biosynthesispathway, such as dihydrodipicolinate synthase, diaminopimelatedecarboxylase, and aspartate semialdehyde dehydrogenase.

For the genes of enzymes that are subject to feedback inhibition byL-lysine, such as dihydropicolinate acid synthase and aspartokinase, itis desirable to reduce or eliminate feedback inhibition by L-Lysine.

Furthermore, the ability to produce L-amino acid can also be enhanced byincreasing the activity of proteins involved in the secretion of L-aminoacids to the outside of the bacterium. For example, the LysE proteinencoded by the lysE gene is known to contribute to the secretion ofL-lysine to the outside of the cell (Vrljic, M., Sahm, H. and Eggeling,L. (1996) Mol. Microbiol. 22, 815-826, International Publication No.97/23597). The wild-type of the lysE gene derived from bacteria of thegenus Brevibacterium does not function at all in bacteria of the generaMethylophilus and Methylobacillus, but by mutating lysE, this protein isable to function in methylotrophes. The mutant lysE24 protein describedbelow is an example of such a mutant lysE protein. Themethanol-assimilating bacterium AJ110196, which has the lysE24 gene aswell as the gene encoding dihydrodipicolinate synthase modified so it isnot subject to feedback inhibition by L-lysine, was deposited (FERMBP-10434) under the Budapest Treaty at the International Patent OrganismDepositary (Chuo No. 6, 1-banchi, 1-chome, Tsukuba-shi Higashi,Ibaraki-ken, Japan, Postal Code 305-8566) of the National Institute ofAdvanced Industrial Science and Technology, an IndependentAdministrative Institution, on Oct. 12, 2005.

The genes that can be employed in microorganisms will be specificallydescribed below.

The dapA* Gene

The DNA encoding dihydrodipicolinate synthase which is desensitized tofeedback inhibition by L-lysine (hereinafter, “the dapA* gene”) is notparticularly limited, however, it is preferably, for example, the DNAencoding a dihydrodipicolinate synthase derived from, or native to, abacterium belonging to the genus Escherichia, and having a mutation todesensitize feedback inhibition by L-lysine.

An example of the DNA encoding wild-type dihydrodipicolinate synthasederived from, or native to, a bacterium belonging to the genusEscherichia is that which encodes the amino acid sequence of SEQ ID NO:41. An example of the mutation which results in desensitization of thefeedback inhibition by L-lysine is the histidine residue at position 118in the amino acid sequence of SEQ ID NO: 41 is replaced with a tyrosineresidue (H118Y mutation). Therefore, an example of the dapA* gene is theDNA encoding the amino acid sequence of SEQ ID NO: 41 in which thehistidine residue at position 118 is replaced with a tyrosine residue.

The dapA* gene may also be a DNA encoding a protein which is at least80%, preferably at least 90%, more preferably at least 95%, particularlypreferably at least 98% homologous to the entire amino acid sequence ofSEQ ID NO: 41, and which also has the H118Y mutation anddihydrodipicolinate synthase activity.

Moreover, the dapA* gene may encode a protein having the amino acidsequence of SEQ ID NO: 41, but which includes substitution, deletion,insertion, addition, or the like of one or several amino acids, so longas the protein has the H118Y mutation and the dihydrodipicolinatesynthase activity is not impaired.

Although the number of amino acids which constitutes “several” maydiffer depending on their relative positions in the three-dimensionalstructure of the protein, or the types of amino acid residues beingaltered, it is specifically 1 to 20, preferably 1 to 10, and morepreferably 1 to 5. The above-mentioned substitution of amino acids ispreferably a conservative substitution. Examples of conservativesubstitutions include: substitution of ser or thr for ala; substitutionof gln, his, or lys for arg; substitution of glu, gln, lys, his, or aspfor asn; substitution of asn, glu, or gln for asp; substitution of seror ala for cys; substitution of asn, glu, lys, his, asp, or arg for gln;substitution of gly, asn, gln, lys, or asp for glu; substitution of profor gly; substitution of asn, lys, gln, arg, or tyr for his;substitution of leu, met, val, or phe for ile; substitution of ile, met,val, or phe for leu; substitution of asn, glu, gln, his, or arg for lys;substitution of ile, leu, val or phe for met; substitution of trp, tyr,met, ile, or leu for phe; substitution of thr or ala for ser;substitution of ser or ala for thr; substitution of phe or tyr for trp;substitution of his, phe, or trp for tyr; and substitution of met, ile,or leu for val. The amino acid substitutions, deletions, insertions,additions, inversions, or the like may be the result of anaturally-occurring mutation (mutant or variant) due to an individualdifference, or a difference in the bacterial species harboring thedihydrodipicolinate synthase gene.

The dapA* gene may be able to hybridize with a complementary strand ofthe nucleotide sequence of SEQ ID NO: 40, or a probe that can beprepared from the sequence, under stringent conditions so long as thegene encodes a protein having the H118Y mutation and dihydrodipicolinatesynthase activity. The term “stringent conditions” refers to conditionswhere a so-called specific hybrid is formed and a non-specific hybrid isnot formed. It is difficult to clearly define the conditions with anumerical value, but examples include conditions corresponding to a saltconcentration and temperature of washing which are typical for astandard Southern hybridization, e.g., washing at 60° C. with 1×SSC and0.1% SDS, preferably at 60° C. with 0.1×SSC and 0.1% SDS, and morepreferably at 68° C. with 0.1×SSC and 0.1% SDS, once or preferably twiceor three times.

The dapA* gene may be obtained by site-specific mutagenesis, or from theRSFD80 plasmid as described below.

It is known that the wild-type dihydrodipicolinate synthase derived fromcoryneform bacteria is not subject to feedback inhibition by L-lysine (JGen Microbiol. 1988 December; 134 (12):3221-9.). Therefore, it is notalways necessary to use a DNA encoding a dihydrodipicolinate synthasewhich is desensitized to feedback inhibition by L-lysine.

DNA encoding mutant LysE that promotes export or secretion of L-Lysineto the outside of the bacterium when the DNA is introduced intomethanol-assimilating bacterium

Examples of DNA encoding a mutant LysE protein that promotes export ofL-lysine to the outside of a bacterium when the DNA is introduced into amethanol-assimilating bacterium include the LysE24 gene (US2003-0124687), lysE56 gene (US 2004-0146974), and lysE24 m5 (WO2006/059715).

The expression “promoting export or secretion of L-lysine to the outsideof a bacterium” means that when a methanol-assimilating bacteriumcontaining the DNA is cultured in a medium, the amount of L-lysineexported into the medium increases as compared with themethanol-assimilating bacterium not containing the DNA. An increase inthe export of the L-lysine to the outside of the cell occurs when thereis an increase in L-lysine accumulation in the medium during the cultureof the methanol-assimilating bacterium containing the DNA as comparedwith the accumulation when methanol-assimilating bacterium notcontaining the DNA is cultured.

The DNA encoding mutant LysE that promotes export of L-lysine to theoutside of a bacterium when the DNA is introduced into themethanol-assimilating bacterium is preferably, but is not limited to, aDNA encoding the LysE protein derived from a bacterium belonging to thegenus Brevibacterium and having a mutation which promotes the export ofL-lysine to the outside of a bacterium when the DNA is introduced intothe methanol-assimilating bacterium, and examples thereof include theLysE24 gene (US 2003-0124687), lysE56 gene (US 2004-0146974), and lysE24m5 (WO 2006/059715).

(1) lysE24 Gene

The wild-type lysE gene encodes a protein (wild-type lysE protein: SEQID NO: 55) having a loop region and six hydrophobic helixes that isinvolved in the export of L-lysine to the outside of a bacterium. Themutant lysE24 gene encodes a mutant lysE protein that does not have theloop region and promotes export of L-lysine, L-arginine, or both whenthe DNA is introduced into the methanol-assimilating bacterium. Examplesof the LysE24 gene include the LysE24 gene described in JP 2004-166594 A(US 2005-003495).

An example includes the DNA encoding the protein of SEQ ID NO: 51. Inaddition, the protein may be not less than 80%, preferably not less than90%, more preferably not less than 95%, particularly preferably not lessthan 98% homologous to the entire amino acid sequence of SEQ ID NO: 51,so long as the gene can promote export of L-lysine to the outside of abacterium when it is introduced into a methanol-assimilating bacterium.

Moreover, the gene may be the DNA encoding the protein having thesequence of SEQ ID NO: 51, but which includes substitutions, deletions,insertions, additions, or the like of one or several amino acids so longas the activity for promoting export of L-lysine to the outside of acell is not impaired.

Moreover, the lysE24 gene may be a DNA which is able to hybridize with acomplementary strand of the nucleotide sequence of SEQ ID NO: 50, or aprobe that can be prepared from the nucleotide sequence, under stringentconditions so long as the gene encodes a protein having the activity forpromoting export of L-lysine to the outside of a cell.

The definitions of the terms “several” and “stringent conditions” andpreferable amino acid substitutions are the same as described above.

The lysE24 gene can be obtained from, for example, the plasmid pRSlysE24described in JP 2004-166594 A (US 2005-003495). The E. coli JM109 straintransformed with pRSlysE24 was designated as AJ13830, and deposited atthe National Institute of Advanced Industrial Science and Technology,International Patent Organism Depository on Jun. 4, 2001 and given anaccession number of FERM P-18369. Then, the deposit was converted to aninternational deposit under the provisions of the Budapest Treaty on May13, 2002, and given the accession number FERM BP-8040.

(2) lysE56 Gene

An example of the lysE56 gene is the gene encoding the protein havingthe amino acid sequence of SEQ ID NO: 55 in which at least the glycineresidue at position 56 is replaced with another amino acid residue (US2004-0146974). The gene may also encode a protein having the amino acidsequence of SEQ ID NO: 55 but which is not less than 80%, preferably notless than 90%, more preferably not less than 95%, particularlypreferably not less than 98% homologous to SEQ ID NO: 55, and in whichthe glycine residue at position 56 is replaced with another amino acidresidue, so long as the gene can promote export of L-lysine to theoutside of a methanol-assimilating bacterium.

(3) lysE24 m5 Gene

This gene includes a DNA having the nucleotide sequence of the lysE24gene, but which has been modified so that each reading frame includes astop codon, and promotes export of L-lysine, L-arginine, or both to theoutside of a methanol-assimilating bacterium when the DNA is introducedinto the bacterium. Specific examples include the DNA having thenucleotide sequence of SEQ ID NO: 56 (WO 2006/059715). The gene may be aDNA which is able to hybridize with a complementary strand of thenucleotide sequence of SEQ ID NO: 56, or a probe that can be preparedfrom the nucleotide sequence, under stringent conditions so long as thegene encodes a protein having the activity for promoting export ofL-lysine to the outside of a cell.

The ddh Gene

The diaminopimelate dehydrogenase activity can be enhanced using a geneencoding diaminopimelate dehydrogenase (hereinafter, “the ddh gene”).

An example of the ddh gene includes, but is not limited to, a DNAencoding diaminopimelate dehydrogenase derived from, or native to, acoryneform bacterium (SEQ ID NO: 53).

The ddh gene may be a DNA encoding a protein which is not less than 80%,preferably not less than 90%, more preferably not less than 95%,particularly preferably not less than 98% homologous to the entire aminoacid sequence of SEQ ID NO: 53, and which has diaminopimelatedehydrogenase activity.

The ddh gene may also be a DNA encoding the protein having the sequenceof SEQ ID NO: 53, but which includes one or several amino acidsubstitutions, deletions, insertions, additions, or the like, so long asdiaminopimelate dehydrogenase activity is not impaired.

Moreover, the ddh gene may be a DNA which is able to hybridize with acomplementary strand of the nucleotide sequence of SEQ ID NO: 52, or aprobe that can be prepared from the nucleotide sequence, under stringentconditions so long as the gene encodes a protein having diaminopimelatedehydrogenase activity.

The definitions of the terms “several” and “stringent conditions” andpreferable amino acid substitution are the same as described above.

The ddh gene from coryneform bacterium can be obtained by amplificationthrough PCR using two oligonucleotide primers (for example, SEQ ID NOS:11 and 12 described in WO/9516042, U.S. Pat. No. 6,040,160), which areprepared based on the known nucleotide sequence of ddh fromCorynebacterium glutamicum (Ishino, S. et al., Nucleic Acid Res., 15,3917 (1987)) and using the chromosomal DNA of Brevibacteriumlactofermentum or Corynebacterium glutamicum as the template.

The lysA Gene

The diaminopimelate decarboxylase activity can be enhanced using thediaminopimelate decarboxylase gene (hereinafter, “the lysA gene”). Anexample of the lysA gene includes, but is not limited to, the DNAencoding diaminopimelate decarboxylase derived from, or native to, abacterium belonging to the genus Methylophilus (SEQ ID NO: 49).

The lysA gene may be a DNA encoding a protein which is not less than80%, preferably not less than 90%, more preferably not less than 95%,particularly preferably not less than 98% homologous to the entire aminoacid sequence of SEQ ID NO: 49 and having diaminopimelate decarboxylaseactivity.

The lysA gene may also be a DNA encoding a protein having the sequenceof SEQ ID NO: 49, but which includes one or several amino acidsubstitutions, deletions, insertions, additions, or the like so long asdiaminopimelate decarboxylase activity is not impaired.

Moreover, the lysA gene may be a DNA which is able to hybridize with acomplementary strand of the nucleotide sequence of SEQ ID NO: 48, or aprobe that can be prepared from the nucleotide sequence, under stringentconditions so long as the gene encodes a protein having diaminopimelatedecarboxylase activity.

The definitions of the terms “several” and “stringent conditions” andpreferable amino acid substitutions are the same as described above.

The lysA gene of Methylophilus methylotrophus can be obtained by PCRusing two oligonucleotide primers prepared based on the known sequenceand using the chromosomal DNA of Methylophilus methylotrophus as thetemplate.

The dapB Gene

The dihydrodipicolinate reductase activity can be enhanced using a geneencoding dihydrodipicolinate reductase (hereinafter, “the dapB gene”).An example of the dapB gene includes, but is not limited to, a DNAencoding dihydrodipicolinate reductase derived from, or native to, abacterium belonging to the genus Escherichia (SEQ ID NO: 43).

The dapB gene may be a DNA encoding a protein which is not less than80%, preferably not less than 90%, more preferably not less than 95%,particularly preferably not less than 98% homologous to the entire aminoacid sequence of SEQ ID NO: 43 and having dihydrodipicolinate reductaseactivity.

The dapB gene may also be a DNA encoding the protein having a sequenceof SEQ ID NO: 43, but which includes one or several amino acidsubstitutions, deletions, insertions, additions, so long as thedihydrodipicolinate reductase activity is not impaired.

Moreover, the dapB gene may be a DNA which is able to hybridize with acomplementary strand of the nucleotide sequence of SEQ ID NO: 42, or aprobe that can be prepared from the nucleotide sequence, under stringentconditions so long as the gene encodes a protein having thedihydrodipicolinate reductase activity.

The definitions of the terms “several” and “stringent conditions” andpreferable amino acid substitutions are the same as described above.

The dihydrodipicolinate reductase gene (dapB) can be amplified by PCRusing two oligonucleotide primers prepared based on the known nucleotidesequence and using the chromosomal DNA of E. coli as the template.

The asd Gene

The aspartate-semialdehyde dehydrogenase activity can be enhanced usingthe gene encoding aspartate-semialdehyde dehydrogenase (hereinafter,“the asd gene”). An example of the asd gene includes, but is not limitedto, a DNA encoding aspartate-semialdehyde dehydrogenase derived from, ornative to, a bacterium belonging to the genus Escherichia (SEQ ID NO:45).

The asd gene may be a DNA encoding a protein which is not less than 80%,preferably not less than 90%, more preferably not less than 95%,particularly preferably not less than 98% homologous to the entire aminoacid sequence of SEQ ID NO: 45 and having aspartate-semialdehydedehydrogenase activity.

The asd gene may also be a DNA encoding a protein having the sequence ofSEQ ID NO: 45, but which includes one or several amino acidsubstitutions, deletions, insertions, additions, or the like so long asthe aspartate-semialdehyde dehydrogenase activity is not impaired.

Moreover, the asd gene may be a DNA which is able to hybridize with acomplementary strand of the nucleotide sequence of SEQ ID NO: 44, or aprobe that can be prepared from the nucleotide sequence, under stringentconditions so long as the gene encodes a protein havingaspartate-semialdehyde dehydrogenase activity.

The definitions of the terms “several” and “stringent conditions” andpreferable amino acid substitutions are the same as described above.

The aspartate-semialdehyde dehydrogenase gene (asd) can be amplified byPCR using two oligonucleotide primers prepared based on the knownnucleotide sequence and using the chromosomal DNA of E. coli as thetemplate.

The lysC* Gene

The methanol-assimilating bacterium may further include a DNA encodingaspartokinase that is desensitized to feedback inhibition by L-lysine.

Examples of the DNA encoding aspartokinase that is desensitized tofeedback inhibition by L-lysine (hereinafter, “the lysC* gene”)preferably include, but are not limited to, the DNA encodingaspartokinase derived from, or native to, a bacterium belonging to thegenus Escherichia and having a mutation to desensitize to feedbackinhibition by L-lysine.

An example of the DNA encoding the wild-type aspartokinase derived froma bacterium belonging to the genus Escherichia is the DNA encoding theamino acid sequence of SEQ ID NO: 47. An example of the mutation todesensitize feedback inhibition by L-lysine is the threonine residue atposition 352 is replaced with an isoleucine residue in the amino acidsequence of SEQ ID NO: 47 (T352I mutation). Therefore, an example of thelysC* gene is the DNA encoding the amino acid sequence of SEQ ID NO: 47in which the threonine residue at position 352 is replaced with anisoleucine residue.

The lysC* gene may be a DNA encoding a protein which is not less than80%, preferably not less than 90%, more preferably not less than 95%,particularly preferably not less than 98% homologous to the entire aminoacid sequence of SEQ ID NO: 47, includes the T352I mutation, and hasaspartokinase activity.

The lysC*gene may also be a DNA encoding the protein having the sequenceof SEQ ID NO: 47, but which includes one or more amino acidsubstitutions, deletions, insertions, additions, or the like so long asit includes the T352I mutation and the aspartokinase activity is notimpaired.

Moreover, the lysC* gene may be a DNA which is able to hybridize with acomplementary strand of the nucleotide sequence of SEQ ID NO: 46, or aprobe that can be prepared from the nucleotide sequence, under stringentconditions so long as the gene has the T352I mutation and encodes aprotein having aspartokinase activity.

The definitions of the terms “several” and “stringent conditions” andpreferable amino acid substitutions are the same as described above.

The lysC* gene may be obtained by site-specific mutagenesis, or from theRSFD80 plasmid as described below.

The DNA encoding aspartokinase which is desensitized to feedbackinhibition by L-lysine does not necessarily have to be DNA encodingvariant aspartokinase. That is, the wild-type form can be employed solong as it is not subject to L-lysine feedback inhibition.

In the present invention, the phrase “increasing the activity of enzyme”means that the enzymatic activity in the cell is increased relative tothe activity in the wild-type strain (for example, the M. methylotrophusAS 1 strain) or the parent strain (for example, a cell with nomodifications to increase the specific combination of enzymes asdescribed herein), and also means that the bacterium exhibits anenzymatic activity that is not present in the wild-type or parentstrain. The methods of measuring the activity of the above-describedenzymes are known, and increasing the activity in the cell can bereadily confirmed by those of ordinary skill in the art.

Examples of the procedure which can be used to enhance or increase theintracellular activity include, but are not limited to, the followingprocedures and combinations thereof:

(1) Transformation with a plasmid carrying the DNA encoding eachprotein.

(2) Incorporation into the chromosome of the DNA encoding the eachprotein.

(3) Modifying the promoter sequences of the genes encoding the proteins.

The methanol-assimilating bacterium should have the ability to produceL-lysine as a result of the modifications mentioned above, for example,increasing the expression of one or more of the following genes: lysE24,dapA*, lysA, ddh, dapB, asd, (lysC*). The phrase “the ability to produceL-lysine” indicates the ability to produce a recoverable quantity ofL-lysine in the medium when the methanol-assimilating bacterium iscultivated in medium.

The methanol-assimilating bacterium may have mutations which makes itauxotrophic, analog-resistant, and/or metabolically controlled, or thelike, and then the modifications such as those described above may bemade. Examples include bacteria that have been modified as set forthabove, such as variants requiring L-homoserine, L-threonine, and/orL-methionine (JP48-28078A and 56-6499A); mutants which are auxotrophicfor inositol or acetic acid (JP55-9784A and 56-8692A); and mutants whichare resistant to oxalidine, lysine hydroxamate,S-(2-aminoethyl)cysteine, γ-methyl lysine, α-chlorocaprolactam,DL-α-amino-ε-caprolactam, α-aminolauryllactam, aspartic acid-analog,sulfa agents, quinoids, or N-lauroyl leucine.

Alternatively, mutations resulting in auxotrophic mutants, analogresistance, metabolism controls, and the like can be introduced afterimparting the above-described modification (gene amplification).

Examples of methods for increasing expression of the aspartokinase gene(lysC* gene) and dihydrodipicolinate synthase gene (dapA* gene) that isnot subject to feedback inhibition by L-lysine and will be shown below.

To increase the expression of the dapA* gene and the lysC* gene, DNAfragments of the genes are ligated to a vector that is able to replicatein bacteria of the genus Methylophilus, desirably a multicopy vector, toprepare recombinant DNA. This is then transformed into the Methylophilusbacterium host. Since the number of copies of these genes is increased,the activity of dihydrodipicolinate synthase and aspartokinase in thecell is increased. Dihydrodipicolinate synthase is abbreviated to“DDPS,” aspartokinase to “AK,” and aspartokinase III to “AKIII.”

Any microorganism of the genus Methylophilus having DNA capable ofexpressing DDPS activity and AK activity can be used. The microorganismmay be a wild-type strain or a mutant strain derived from a wild-typestrain. E. coli (Escherichia coli) K-12 strain and Methylophilusmethylotrophus ASI strain (NCIMB 10515) may be used as the host strain.The DNA sequence of the gene encoding DDPS derived from genusEscherichia (Richaud, F, et al. J. Bacteriol., 297 (1986)) and the geneencoding AKIII (Cassan, M., Pasrot, C., Cohen, G. N. and Patte, J. C.,J. Biol. Chem., 261, 1052 (1986)) have both been determined. Thus, thesegenes can be obtained by primers synthesized based on the sequences ofthe genes and using PCR with chromosomal DNA of E. coli K-12 or the likeas the template. Examples of dapA and lysC derived from E. coli areshown below, but the genes are not limited to these.

The DDPS and AK should not be subject to feedback inhibition byL-lysine. Wild-type DDPS derived from E. coli is known to be subject tofeedback inhibition by L-lysine. Wild-type AKIII derived from E. coli isalso known to be inhibited by L-lysine and to be subject to feedbackinhibition by L-lysine. Accordingly, mutations that remove this feedbackinhibition are desirably introduced prior to introduction into bacteriaof the genus Methylophilus.

However, for example, since DDPS derived from bacteria of the genusCorynebacterium is not subject to feedback inhibition by L-lysine, theDDPS gene and the AK gene are not necessarily the above-noted mutantgenes.

It is possible to obtain dapA* encoding DDPS and lysC* encoding AK whichare not subject to feedback inhibition by performing PCR with twooligonucleotide primers prepared using known sequences employing plasmidcontaining these genes as the template.

The broad-host-range plasmid RSFD80 is known to contain dapA* and lysC*(WO95/16042). An E. coli JM109 strain that has been transformed withthis plasmid is named “AJ12396.” This strain was deposited (FERMP-13936) with the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, MITI (now the InternationalPatent Organism Depositary of the National Institute of AdvancedIndustrial Science and Technology, an Independent AdministrativeInstitution) on Oct. 28, 1993, and converted to international depositunder the Budapest Treaty on Nov. 1, 1994, and given the designationFERM BP-4859. RSFD80 can be obtained by known methods from the AJ12396strain.

The sequence of the dapA* on the RSFD80 has a T instead of a C at baseposition 597 (see the wild-type dapA gene at SEQ ID NO: 40). Thus, thismutation results in the encoded protein sequence having a tyrosineresidue instead of a histidine residue at position 118 in the amino acidsequence of SEQ ID NO: 41. The sequence of the lysC* on the RSFD80 has aT instead of a C at base position 1638 (see the wild-type lysC of SEQ IDNO: 46). Thus, this mutation results in the encoded protein sequencehaving an isoleucine residue instead of the threonine residue atposition 352 in the amino acid sequence of SEQ ID NO: 47.

The plasmid used to clone the gene can be able to replicate in amicroorganism such as Escherichia; examples are pBR322, pTWV228, pMW119,and pUC19.

The vector which is able to function in a bacterium of the genusMethylophilus is, for example, a plasmid that is autonomously replicablein a bacterium of the genus Methylophilus. Examples include thebroad-host-range vector RSF1010 and its derivatives, such as pAYC32(Chistorerdov, A. Y., Tsygankov, Y. D. Plasmid, 1986, 16, 161-167),pMFY42 (Gene, 44, 53 (1990)), pRP301, and pTB70 (Nature, 287, 396(1980)). pBBR1, another broad-host-range vector that is incompatiblewith RSF1010, and its derivatives, such as pBHR1, (Antoine, R. andLocht, C., Molecular Microbiology, 6, 1785-99 (1992)) are furtherexamples.

When the DNA encoding dapA*, lysC*, and other proteins is ligated to thevector which is able to function in the Methylophilus bacterium, thevector is cut with restriction enzymes corresponding to the terminals ofthe DNA fragments containing these genes. The ligation is normallyconducted with a ligase such as T4 DNA ligase. These genes can belocated on separate vectors, or located on a single vector.

Methods well known to those of ordinary skill in the art can be employedto cut and ligate the DNA, prepare chromosomal DNA, conduct PCR, prepareplasmid DNA, conduct transformation, identify oligonucleotides for useas primers, and the like. These methods are described in Sambrook, J.,Fritsch, E. F., and Maniatis, T., “Molecular Cloning, A LaboratoryManual, Second Edition,” Cold Spring Harbor Laboratory Press (1989) andthe like.

Any method that allows for adequate transformation efficiency can beemployed to introduce the recombinant DNA thus prepared into theMethylophilus bacterium. One example is electroporation (CanadianJournal of Microbiology, 43. 197 (1997)).

DDPS and AK activity can be increased by introducing multiple copies ofdapA* and lysC* into the chromosomal DNA of the Methylophilus bacterium.Introduction of multiple copies of the DNA can be accomplished by usinga sequence that is present in multiple copies on the chromosomal DNA asa target, and conducting homologous recombination. Repetitive DNA andthe inverted repeats present on the ends of transposons are typicallypresent in multiple copies on the chromosomal DNA. Alternatively, as isdisclosed in JP2-109985A, dapA* and/or lysC* can be carried on atransposon, and transposed resulting in the introduction of multiplecopies into the chromosomal DNA. Regardless of the chosen, the increasein the number of copies of dapA* and lysC* in the transformant amplifiesthe DDPS and AK activity.

In addition to amplifying the genes as described above, DDPS and AKactivity can also be increased by substituting the native promoters ofdapA* and lysC* with a stronger promoter (see Japanese PatentApplication Publication No. Heisei 1-215280). Examples of known strongpromoters are the lac promoter, trp promoter, trc promoter, tacpromoter, lambda phage PR promoter, PL promoter, tet promoter, amyEpromoter, and spac promoter. Replacing the native promoter with one ofthese promoters increases the expression of dapA* and lysC*, therebyamplifying DDPS and AK activity. The enhancement of expressionregulation sequences can be combined with increasing the number ofcopies of dapA* and lysC*.

Enhancing the expression of the dapA* gene and lysC* gene to increasethe activity of DDPS and AK within the cell has been described.Increasing the expression of the lysE24 gene, dapB gene, lysA gene, ddhgene, and asd gene is also similarly possible.

In addition to enhancing the expression of the above-described six orseven genes, other enzymes involved in the biosynthesis of L-lysine canalso be increased in the methanol-assimilating bacterium. Examples ofsuch enzymes include phosphenol pyruvate carboxylase (JP 60-87788A),aspartate aminotransferase (JP6-102028A), enzymes in the diaminopimelicacid pathway, such as diaminopimelate epimerase (JP2003-135066A), andenzymes in the aminoadipic acid pathway, such as homoaconitatehydratase.

Examples of enzymes that catalyze reactions directed away from theL-lysine synthesis pathways to produce compounds other than L-lysine arehomoserine dehydrogenase (see WO 95/23864) and L-lysine decarboxylase(Japanese Patent Application Publication No. 2004-254544). Modificationscan be made in these enzymes so that enzymatic activity is reduced byinactivation of the gene by homologous recombination.

The methanol-assimilating bacterium can also be modified so thatL-methionine is required for growth (JP2004-248669A) by subjecting thebacterium to natural mutation or a mutagenic treatment so that it cannotgrow in a medium that does not contain L-methionine, or by disruption ofthe metA gene (JP2004-248669A) or metF gene (SEQ ID NO: 58).

Examples of bacteria producing L-amino acids other than L-lysine aredescribed below. The ability to produce an L-amino acid can be impartedby obtaining a variant with an auxotrophic mutant, an analog-resistantmutant, or a metabolically controlled mutant; creating a strain whichoverexpresses an enzyme involved in the biosynthesis of the L-aminoacid; or applying a method that has conventionally been used to breedbacteria of the genus Escherichia, coryneform bacteria, or the like (seeAmino Acid Fermentation, Gakkai Shuppan Center, 1st ed. May 30, 1986,pp. 77-100).

Furthermore, L-amino acid-producing bacteria can also be constructed byincreasing the expression of genes related to the biosynthesis of theL-amino acid and attenuating the genes that degrade the L-amino acid.

2-2 Imparting the Ability to Produce L-Glutamic Acid

The ability to produce L-glutamic acid can be imparted to aMethylophilus bacterium, for example, by introducing DNA encodingenzymes such as glutamate dehydrogenase (JP61-268185A), glutaminesynthetase, glutamate synthase, isocitrate dehydrogenase (JP62-166890and 63-214189), aconitate hydratase (JP62-294086A), citrate synthase(JP62-201585A and Showa 63-119688A), phosphenol pyruvate carboxylase(JP60-87788A and JP62-55089A), pyruvate dehydrogenase, pyruvate kinase,phosphenol pyruvate synthase, enolase, phosphoglyceromutase,phosphoglycerate kinase, glyceryl aldehyde-3-phosphate dehydrogenase,triosephosphate isomerase, fructose bisphosphate aldolase,phosphofructokinase (JP63-102692A), glucose phosphate isomerase, andglutamine oxoglutarate aminotransferase (WO 99/07853).

The activity of enzymes that catalyze reactions which result indegradation of L-glutamic acid, or which direct reactions away from thebiosynthesis pathway of L-glutamic acid so that compounds other thanL-glutamic acid are produced, can be decreased or deleted. Examples ofthese enzymes include α-ketoglutarate dehydrogenase (αKGDH), isocitratelyase, phosphate acetyl transferase, acetate kinase, acetohydroxylatesynthase, acetolactate synthase, formate acetyltransferase, lactatedehydrogenase, glutamate decarboxylase, and 1-pyrroline dehydrogenase.

2-3 Imparting the Ability to Produce L-Threonine

The ability to produce L-threonine can be imparted or increased by, forexample, increasing the activities of aspartokinase, homoserinedehydrogenase, homoserine kinase, and threonine synthase. The activityof these enzymes can be increased, for example, by transforming aMethylophilus bacterium with a recombinant plasmid containing thethreonine operon (see JP 55-131397A, JP59-31691A, JP56-15696A, andJP3-501682A).

Furthermore, the ability to produce L-threonine can be imparted andincreased by amplifying or introducing the threonine operon whichincludes the gene encoding aspartokinase which is not subject tofeedback inhibition by L-threonine (JP1-29559A), the gene encodinghomoserine dehydrogenase (JP60-012995A), or the genes encodinghomoserine kinase and homoserine dehydrogenase (JP61-195695).

The ability to produce L-threonine can also be enhanced by introducingDNA encoding a mutant phosphenol pyruvate carboxylase which is notsubject to feedback inhibition by aspartic acid.

2-4 Imparting the Ability to Produce L-Valine

The ability to produce L-valine can be imparted by, for example,introducing genes related to the biosynthesis of L-valine from which thecontrol mechanisms have essentially been removed. Mutations thatessentially remove the control mechanisms from genes related to thebiosynthesis of L-valine can also be introduced.

The ilvGMEDA operon of E. coli, for example, is related to thebiosynthesis of L-valine. The threonine deaminase encoded by the ilvAgene catalyzes the deamination reaction from L-threonine to2-ketobutyric acid, which is a rate-limiting step in the biosynthesis ofL-isoleucine. Accordingly, for the L-valine synthesis reaction toprogress efficiently, an operon that does not express threoninedeaminase activity should be used. Therefore, the ilvA gene on theilvGMEDA operon should be mutated or partially or entirely deleted sothat threonine deaminase activity is not expressed.

Furthermore, expression of the ilvGMEDA operon is regulated andattenuated by L-valine, L-isoleucine, and/or L-leucine. Thus, the regionrequired for attenuation should be deleted or mutation so thatexpression is not inhibited by the produced L-valine.

The ilvGMEDA operon that does not express threonine deaminase activityand that is not attenuated by the produced L-amino acid can be obtainedby subjecting the wild-type ilvGMEDA operon to a mutagenic treatment orthrough changes caused by genetic recombination techniques (see WO96/06926).

2-5 Imparting the Ability to Produce L-Leucine

The ability to produce L-leucine can be imparted or increased by, forexample, introducing genes relating to the biosynthesis of L-leucinefrom which the control mechanisms have been essentially removed into theMethylophilus bacteria, in addition to the above-described propertiesrequired for the production of L-valine. It is also possible tointroduce mutations that essentially remove the control mechanisms ofthe genes related to the biosynthesis of L-leucine. An example of such agene is the leuA gene which has been mutated to remove the inhibition byL-leucine.

2-6 Imparting the Ability to Produce L-Isoleucine

The ability to produce L-isoleucine can be imparted by introducing thethrABC operon which includes the E. coli thrA gene encodingaspartokinase I-homoserine dehydrogenase I which has been mutated toremove the inhibition by L-threonine, and the ilvGMEDA operon, which hasbeen mutated to delete regions required for attenuation. The operon mayalso contain the ilvA gene encoding threonine deaminase which has beenmutated to delete the inhibition by L-isoleucine (see JP8-47397A).

2-7 Imparting the Ability to Produce Other Amino Acids

The biosynthesis of L-tryptophan, L-phenylalanine, L-tyrosine,L-threonine, and L-isoleucine can be increased by enhancing the abilityto produce phosphenol pyruvate in the Methylophilus bacteria (WO97/08333).

The ability to produce L-phenylalanine and L-tyrosine can be enhanced byamplifying or introducing the desensitizable chorismatemutase-prephenate dehydratase (CM-PDT) gene (see JP62-130693A) or thedesensitizable 3-deoxy-D-arabino-hepturonic acid-7-phosphate synthase(DS) gene (see JP5-236947A and JP61-124375A).

Furthermore, the ability to produce L-tryptophan can be enhanced byamplifying or introducing the tryptophan operon containing the geneencoding desensitizable anthranylate synthase (JP57-71397A andJP62-244382A, and U.S. Pat. No. 4,371,614).

The phrase “enzymatic activity is increased” indicates that the activityof the enzyme in the cell is higher than in the wild-type strain. When abacterial strain with increased enzymatic activity is obtained throughmodification(s) made by genetic recombination techniques, then theactivity of the enzyme in the cell is greater than before themodification(s). The phrase “enzymatic activity is decreased” indicatesthat the activity of the enzyme within the cell is lower than in thewild-type strain. When a bacterial strain with reduced enzymaticactivity is obtained through modification(s) made by geneticrecombination techniques, then the activity of the enzyme within thecell is lower than before the modification(s).

EXAMPLES

The present invention is described in detail below through the followingnon-limiting embodiments.

Reference Example 1

Unless specifically stated otherwise, the reagents used in theseexamples were obtained from Wako Pure Chemical Industries, Ltd. orNacalai Tesque, Inc. The compositions of the media are as indicatedbelow. The pH of the media was adjusted with NaOH or HCl.

LB medium: Tryptone peptone (made by Difco) 10 g/L Yeast extract (madeby Difco) 5 g/L NaCl 10 g/L pH 7.0 Steam sterilization was conducted for20 minutes at 120° C. LB agar medium: Bacto agar 15 g/L Steamsterilization was conducted for 20 minutes at 120° C. K₂HPO₄ 1.9 g/LNaH₂PO₄ 1.56 g/L MgSO₄•7H₂O 0.2 g/L (NH₄)₂SO₄ 5 g/L CuSO₄•5H₂O 5 μg/LMnSO₄•5H₂O 25 μg/L ZnSO₄•7H₂O 23 μg/L CaCl₂•2H₂O 72 mg/L FeCl₃•6H₂O 9.7mg/L Sodium pyruvate 2.5 g/L CaCO₃ (made by Kanto 30 g/L Chemical Co.,Inc.) Methanol 2% (vol/vol) pH 7.0

All ingredients except for methanol were steam sterilized for 15 minutesat 121° C. The methanol was added after the components had suitablycooled.

SEII Production Medium:

K₂HPO₄ 1.9 g/L NaH₂PO₄ 1.56 g/L MgSO₄•7H₂O 0.2 g/L (NH₄)₂SO₄ 5 g/LCuSO₄•5H₂O 5 μg/L MnSO₄•5H₂O 25 μg/L ZnSO₄•7H₂O 23 μg/L CaCl₂•2H₂O 72mg/L FeCl₃•6H₂O 9.7 mg/L Sodium pyruvate 2.5 g/L CaCO₃ (made by Kanto 30g/L Chemical Co., Inc.) Methanol 2% (vol/vol) pH 7.0

All ingredients except for methanol were steam sterilized for 15 minutesat 121° C. The methanol was added after the component had suitablycooled.

SEII Agar Medium

K₂HPO₄ 1.9 g/L NaH₂PO₄ 1.56 g/L MgSO₄•7H₂O 0.2 g/L (NH₄)₂SO₄ 5 g/LCuSO₄•5H₂O 5 μg/L MnSO₄•5H₂O 25 μg/L ZnSO₄•7H₂O 23 μg/L CaCl₂•2H₂O 72mg/L FeCl₃•6H₂O 9.7 mg/L Sodium pyruvate 1.0 g/L Methanol 1% (vol/vol)pH 7.0 Bacto agar 15 g/L (made by Difco)

All ingredients except for methanol were steam sterilized for 15 minutesat 121° C. The methanol and, as needed, L-methionine solution adjustedto 20 g/L and sterilized by filtration, were added after the componentshad suitably cooled.

Reference Example 1 Construction of a Mini-Mu System, pMIV-Km,pMIV-Km-EA, pAET7

To increase the number of copies of the lysE24 and variant dapA genes onthe chromosome, the E. coli bacteriophage Mu-phage gene recombinationsystem was employed.

Construction of pMIV5 (FIGS. 1 and 2)

In order to incorporate Mu-phage into the chromosome of Escherichiacoli, a drug resistance gene located between the recognition sequencesattL and attR, and a transferase (Mu transposase) are required. Both ofthese genes do not need to be carried on the same vector. First, thepMIV5 plasmid was contructed, which contains the recognition sequencesattL and attR, and a kanamycin resistance gene, while the pAET7 plasmidwith the Mu transposase was constructed separately. Both of theseplasmids can function when they are present in the same bacterium,resulting in the transfer of the region located between attL and attR tothe chromosomal DNA.

The pMIV5 plasmid was constructed as follows. First, pMW119 (availablefrom TOYOBO Co., Ltd.) is digested with the PvuII restriction enzyme andseparated by agarose gel electrophoresis to collect a fragment of about3.9 kbp. This fragment was ligated with the DNA Ligation Kit (Takara BioInc.), to obtain the pMW1 plasmid. Subsequently, mini-Mu-phage wastransferred to the pMW1 plasmid in E. coli cells. Specifically, pMD4041(Journal of Bacteriology 158, 488-495 (1984)) was introduced into theEscherichia coli K12 strain, and a strain resistant to kanamycin andsensitive to ampicillin was selected to obtain a strain in which theplasmid pMD4041 was eliminated and mini-Mu 4041 was transferred to thechromosome. A factor that represses Mu transfer of mini-Mu4041, forexample, c repressor, has a temperature-sensitive mutation. Therefore,when the strain is cultured at 42° C., c repressor of mini-Mu on thechromosome is deactivated, and the transfer of mini-Mu4041 to thechromosome is significantly activated, resulting in cell death. Thestrain with lysogenized mini-Mu4041 on the chromosome was transformedwith pMW1 at 30° C. This strain was cultured in LB medium until thenumber of cells reached 2×108 cells/ml, and then was treated at 42° C.for 1 hour. In order to obtain a plasmid with mini-Mu4041 transferred tothe pMW1 plasmid, plasmid DNA was prepared from the cells and used totransform an Escherichia coli strain. Plasmids were prepared from 50 ofthe transformants displaying kanamycin and ampicillin resistance, andthe structures of the plasmids were determined by treating with arestriction enzyme, to thereby select the plasmid of interest. Thisplasmid was designated as pMu11. In the plasmid pMu11, mini-Mu4041 wastransferred to the par region in the pMW 1. More specifically, in pMW119 which contains fragments of the known plasmids pBR322 and pSC101,mini-Mu4041 was inserted into position 259, where the boundary positionbetween the plasmids was defined as position 0. The plasmid was digestedwith the HindIII restriction enzyme and separated by agarose gelelectrophoresis to collect a fragment of about 6.9 kbp, and thisfragment was ligated with the DNA Ligation Kit (Takara Bio Inc.), toconstruct the pM12 plasmid (FIG. 1). pM12 was digested withHindIII-AvaIII (EcoT22I) and used as a vector into which the terminatorregion of the PCR-amplified thr operon of E. coli was inserted. PCRamplification was performed using the chromosomal DNA of E. coli as thetemplate and p-ter-thrL-f (SEQ ID NO: 1) and p-ter-thrL-r (SEQ ID NO: 2)as primers, under the following conditions for 25 cycles: denaturationat 94° C. for 20 seconds, annealing at 55° C. for 30 seconds, andextension at 72° C. for 60 seconds. The resulting plasmid was digestedwith the EcoRI-HindIII restriction enzyme, and the PCR-amplifiedmulti-cloning site region and the PCR-amplified ρ-factor independenttranscription termination factor fragment from a bacteriophage fd wereinserted into the plasmid. The multi-cloning site region was amplifiedby PCR using the pUC57 plasmid (Fermentus AB, available from LITHUANIA)as the template and pUC57-MCS-f (SEQ ID NO: 3:) and pUC57-MCS-r (SEQ IDNO: 4) as primers, and PCR was performed under the following conditionsfor 25 cycles: denaturation at 94° C. for 20 seconds, annealing at 55°C. for 30 seconds, and extension at 72° C. for 30 seconds. The amplifiedfragment was digested with EcoRI-BamHI, the recognition sites of whichhad been added to the primer. The ρ-factor independent transcriptiontermination factor fragment was amplified by PCR using the genomic DNAof the bacteriophage fd as the template and ter-fd-f (SEQ ID NO: 5) andter-fd-r (SEQ ID NO: 6:) as primers, and PCR was performed under thefollowing conditions for 25 cycles: denaturation at 94° C. for 20seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for30 seconds. The amplified fragment was digested with EcoRI-HindIII,recognition sites of which had been added to the primer. The threefragments were ligated with the DNA Ligation kit (Takada Bio Inc.) toconstruct the pMIV5 plasmid (FIG. 2). Then, the plasmid was digestedwith EcoRV, and a kanamycin resistance gene fragment obtained bycleaving a commercially-available plasmid (pUC4K) with HincII wasinserted into the plasmid to construct the pMIV5-Km plasmid.

pMIV-Km-lysE24dapA

A gene of interest was inserted into the pMIV5-Km plasmid, and used toincorporate the gene with mini-Mu, and the plasmid was used to transferthe gene fragment of interest to the chromosome. Specifically, theplasmid pMIV5-Km was digested with SmaI, followed by a dephosphorylationtreatment. The fragment lysE24+dapA* was obtained by amplificationthrough PCR using a known plasmid containing the pRSlysEdapA gene (JP2003-61687 A) as the template, and pRS-1s (SEQ ID NO: 8:) and dapA-r(SEQ ID NO: 7:) as primers. PCR was performed under the followingconditions for 25 cycles: denaturation at 94° C. for 20 seconds,annealing at 55° C. for 30 seconds, and extension at 72° C. for 120seconds. PCR-amplified fragments were ligated with the TaKaRa BKL kit(Takara Bio Inc.) to construct the pMIV-Km-lysE24dapA plasmid.

Construction of pAET7 (FIG. 4)

pUC1918 (Gene, (1993) 134, 89-91) was digested with the EcoRIrestriction enzyme, and the resulting fragment was blunt-ended. Thisfragment was used to insert a blunt-ended DNA fragment encoding Mutransposase, and a DNA fragment was obtained by digesting pMu4041(Journal of Bacteriology, (1984), 158, 488-495) with ScaI-Eco47III. Thisplasmid was designated as pUC-MH7. pUC-MH7 was digested with BamHI, andthe resulting DNA fragment encoding Mu transposase was inserted into theBamHI site of pAYC32 (Journal of General Microbiology 137, 169-178(1991)) to obtain the pAET7 plasmid.

Reference Example 2 Incorporation of the lysE24 and Mutant dapA Genesinto the Chromosome of Methylophilus methylotrophus, and the Acquisitionof VAE#1

First, pAET7 was introduced into the M. methylotrophus AS strain byelectroporation, and the bacterium was inoculated onto an SEII platecontaining 50 mg/l streptomycin. Then, the pMIV-Km-lysE24dapA plasmidwas introduced into the resulting transformant to obtain strains thatformed colonies on the SEII plate containing 20 mg/l kanamycin and 50mg/l streptomycin. The mini-Mu cassette includes a kanamycin resistancegene, and the pMIV-Km-lysE24dapA plasmid, which cannot replicate in M.methylotrophus. Therefore, the kanamycin-resistant colonies have themini-Mu cassette inserted into the chromosome. Accordingly, 200 strainswere randomly selected and spread onto an SEII plate containing 50 mg/lstreptomycin and 20 mg/L kanamycin, followed by culturing at 37° C.overnight. Then, bacterial cells present on the medium surface of about0.3 square centimeters were scraped off and inoculated into an SEIIproduction medium (5 ml) containing 50 mg/l streptomycin and 20 mg/Lkanamycin, and the cells were cultured with shaking at 37° C. for 34hours. After completion of the culture, the bacterial cells were removedby centrifugation, and the concentration of L-lysine in each culturesupernatant was measured using a Biotech-analyzer AS-210 (manufacturedby Sakura Seiki Co., Ltd.). The strain containing the highestconcentration of L-lysine was selected and designated as VAE#1.

Reference Example 3 Acquisition of a Strain with High Copy Numbers oflysE24 and Mutant dapA Genes (VAE#8)

The VAE#1 strain was shown to have one or two copies of the mini-Mucassette inserted into the chromosome. Therefore, in order to improveproductivity of L-lysine, the mini-Mu cassette was amplified on thechromosome. The gene encoding the MuC protein, which is capable ofsuppressing Mu transposase activity, is present on the pAET7 plasmidcarrying the Mu transposase. The MuC protein is temperature sensitive,and therefore, when the strain is cultured at 42° C., the Mu transposaseis active, resulting in amplification of the mini-Mu cassette on thechromosome. Specifically, the VAE#1 strain was suspended in SEII liquidmedium to an appropriate concentration, and the suspension was incubatedat 42° C. for 1 hour and diluted to an appropriate concentration. Thebacterial solutions were inoculated onto SEII plates containing 50 mg/Lstreptomycin and 20 mg/L kanamycin to form single colonies. From thesingle colonies, 200 colonies were selected randomly and spread on SEIIplates containing 50 mg/L streptomycin and 20 mg/L kanamycin, followedby culturing at 37° C. overnight. Then, bacterial cells which werepresent on the medium surface of about 0.3 square centimeters werescraped off and inoculated into an SEII production medium (5 ml)containing 50 mg/L streptomycin and 20 mg/L kanamycin, and the cellswere cultured with shaking at 37° C. for 34 hours. After completion ofthe culture, the bacterial cells were removed by centrifugation, and theconcentration of L-lysine in each culture supernatant was measured usinga Biotech-analyzer AS-210 (manufactured by Sakura Seiki Co., Ltd.). Thestrain with the highest concentration of L-lysine was designated asVAE#2. The procedure was repeated 8 times to obtain the VAE#8 strain.The amount of Lys produced by VAE#1 was defined as 100, and the relativevalue of the amount of Lys produced by the VAE#8 strain was calculatedand is shown in Table 1.

TABLE 1 Relative amount of Strain produced Lys (%) VAE#1 100 VAE#8 800

Reference Example 4 Determination of Transfer Site in VAE#8

Next, the site containing the transferred mini-Mu cassette wastransferred onto the chromosome of the VAE#8 strain was determined. Thechromosomal DNA of the VAE#8 strain was prepared and completely digestedwith the SalI restriction enzyme. The resulting fragment was ligated tothe pHSG398 vector, and selection was performed in an LB agar mediumcontaining 12.5 mg/L chloramphenicol and 25 mg/L kanamycin to prepareplasmid DNA from the colonies. There is a kanamycin resistance gene onthe mini-Mu cassette and chromosomal DNA around the transfer site in theplasmid. The nucleotide sequence of the plasmid was determined using asequencing primer (SEQ ID NO: 9), which was designed outwardly in theinside of attR present on the right hand edge of the mini-Mu cassette,to thereby determine the transfer site of the mini-Mu cassette. It isalso possible to construct a strain identical to VAE#8 based on theinformation of the transfer region determined by the above-describedmethod.

Reference Example 5 Imparting Met-Auxotrophy to VAE#8 (#403)

Next, methionine auxotrophy was imparted to the VAE#8 strain. Impartingamino acid auxotrophy to an amino acid-producing bacterium is effectivefor controlling the number of bacterial cells during the culture. TheVAE#8 strain was mutated by treatment with NTG by a known method (WO00/61723, U.S. Pat. No. 7,223,572) and appropriately diluted to a celldensity to form a single colony, and the bacterium was inoculated intoan SEII agar medium containing 0.5 g/L L-methionine. The cells werereplicated on an SEII agar medium containing no L-methionine to obtain astrain that could not grow on the plate, that is, a strain auxotrophicfor L-methionine. The strain was designated as #403. A plurality ofgenes from the #403 strain, which is known to be involved in thebiosynthesis of L-methionine, were cloned by a method well-known to aperson skilled in the art based on the homology to another microorganismto determine the nucleotide sequences. As a result, it was found thatpart of the metF gene encoding 5,10-methylenetetrahydrofolate reductasewas deleted. Specifically, it was found that the region between the 92ndnucleotide and the 344th nucleotide, as counted from the initiationcodon of the metF gene, was deleted. Therefore, L-methionine auxotrophywas imparted by disrupting the metF gene of VAE#8 by a known method (JP2004-229662 A, Homologous recombination method using linear DNA).Details are described in Example 19. The strain with an artificiallydisrupted metF was found to have the same properties as the strainauxotrophic for L-methionine obtained by the above-describedNTG-mutation treatment, #403. A DNA fragment was prepared to disrupt thegene by overlap-PCR (Ho, S, N., Hunt, H. D., Horton, R. M., Pullen, J.K. and Pease, L. R., Gene, 77, 51-9. (1989)). When the #403 strain andthe control strain VAE#8 were cultured in the SEII production mediumcontaining 0.075 g/L L-methionine and 2.5 g/L sodium pyruvate, theamount of Lys increased. The amount of Lys produced by the VAE#8 strainwas defined as 100, and the relative value of the amount of Lys producedby the #403 strain was calculated and is shown in Table 2.

TABLE 2 Relative amount of Strain produced Lys (%) VAE8 100 #403 156

Reference Example 6 Construction of the pMIV-FRTGmFRT andpMIV-FRTGmFRT-EAplasmids (FIG. 3)

In order to further incorporate a mini-Mu cassette into the #403 strain,an insertion cassette having a gene resistant to an antibiotic otherthan kanamycin was constructed. Specifically, the pMIV5 plasmid wasdigested with EcoRV and used as a vector. Then, pKD4 (Proceedings of theNational Academy of Sciences of the United States of America, (2000) 97,6640-6645) was digested with HindIII-NdeI, and the resulting fragmentwas blunt-ended, followed by insertion of the fragment having thekanamycin resistance gene region. This plasmid was designated aspMIV-FRTKmFRT. The plasmid was digested with the BglII restrictionenzyme, and the resulting fragment was blunt-ended, followed byinsertion of the PCR-amplified gentamicin resistance gene fragment. PCRwas performed using pML122 (Gene, 89, 37-46. (1990)) as the template andpGm-f (SEQ ID NO: 10) and pGm-r (SEQ ID NO: 11) as primers, under thefollowing conditions for 25 cycles: denaturation at 94° C. for 20seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for60 seconds. The plasmid was designated as pMIV-FRTGmFRT. A gene ofinterest was inserted into the region between attL and attR to constructa mini-Mu cassette to amplify the gene of interest on the chromosome ofM. methylotrophus (FIG. 3). Specifically, the pMIV-FRTGmFRT plasmid wasdigested with the SmaI restriction enzyme, followed bydephosphorylation. The lysE24+dapA* fragment was obtained byamplification with PCR using a known plasmid having the pRSlysEdapA gene(JP 2003-61687 A, U.S. Pat. No. 7,169,587) as the template, and pRS-1sand dapA-r as primers. PCR was performed under the following conditionsfor 25 cycles: denaturation at 94° C. for 20 seconds, annealing at 55°C. for 30 seconds, and extension at 72° C. for 120 seconds.PCR-amplified fragments were phosphorylated using a TaKaRa BKL kit(Takara Bio Inc.) and ligated to a vector, to thereby construct thepMIV-FRTGmFRT-EA plasmid. The pKD4 and pCP20 plasmids were registered atthe E. coli Genetic Stock Center as CGSC strains #7632 and #7629,respectively, and are available from the Center.

Reference Example 7 Acquisition of a Strain with Higher Copy Numbers ofthe lysE24 and Mutant dapA Genes Using pMIV-Gm-EA (#403-11-Gm)

First, pAET7 was introduced into the #403 strain, and the cells wereinoculated onto an SEII plate containing 50 mg/l streptomycin. Then, thepMIV-FRTGmFRT-EA plasmid was introduced into the resulting pAET7transformant by electroporation to obtain strains that formed colonieson an SEII plate containing 50 mg/l streptomycin. The mini-Mu cassetteincludes a gentamicin resistance gene, and the pMIV-FRTGmFRT-EA plasmidcannot replicate in M. methylotrophus. Therefore, thegentamicin-resistant strain has the mini-Mu cassette inserted into thechromosome. Accordingly, 200 strains were randomly selected and spreadon an SEII plate containing 50 mg/L streptomycin and 20 mg/L gentamicin,followed by culturing at 37° C. overnight. Then, bacterial cells whichwere present on the medium surface of about 0.3 square centimeters werescraped off and inoculated into an SEII production medium (5 ml)containing 50 mg/l streptomycin and 20 mg/L gentamicin, and the cellswere cultured with shaking at 37° C. for 48 hours. After completion ofthe culture, the bacterial cells were removed by centrifugation, and theconcentration of L-lysine in each culture supernatant was measured usinga Biotech-analyzer AS-210 (manufactured by Sakura Seiki Co., Ltd.). Thestrain with the highest concentration of L-lysine was selected anddesignated as #403-11Gm.

Reference Example 8 Determination of the Transfer Site in #403-11-Gm

Next, the site containing the transferred mini-Mu cassette on thechromosome of the #403-11-Gm strain was determined. The chromosomal DNAof the #403-11 strain was prepared and completely digested with the SalIrestriction enzyme. The resulting fragment was ligated to the pHSG398vector, and selection was performed in an LB agar medium containing 12.5mg/L chloramphenicol and 25 mg/L gentamicin to prepare a plasmid DNAfrom the resulting colonies. There is a cloned kanamycin resistance geneon the mini-Mu cassette, as well as the chromosomal DNA around thetransfer site in the plasmid. The nucleotide sequence of the plasmid wasdetermined using a sequencing primer (SEQ ID NO: 12), which was designedin the attR region present on the right hand edge of the mini-Mucassette, to thereby determine the transfer site of the mini-Mucassette. It is also possible to construct a strain identical to#403-11Gm based on the information of the transfer region determined bythe above-described method.

Reference Example 9 Elimination of the Antibiotic-Resistant Marker from#403-11-Gm (pFLP31), and Acquisition of the #403-11 Strain

Construction of pAYCTER3

Synthetic DNAs of SEQ ID NO: 13 and SEQ ID NO: 14, designed to containthe pUC19 multi-cloning site, were annealed by a well-known method toproduce a polylinker. The polylinker was designed to have the sameterminal as that of the fragment cleaved with restriction enzymes EcoRIand BglII. Moreover, the primers of SEQ ID NO: 15 and SEQ ID NO: 16 weresynthesized, and the region encoding the rrnB terminator sequence wasamplified by PCR from the chromosomal DNA of the Escherichia coli K-12strain prepared by a conventional method (Saito and Miura [Biochim.Biophys. Acta, 72, 619 (1963)]). The primers of SEQ ID NO: 13 and SEQ IDNO: 14 were designed to have the recognition sequences of therestriction enzymes BglII and MI, respectively. PCR was performed usingPyrobest DNA polymerase (manufactured by Takara Bio Inc.) under reactionconditions recommended by the manufacturer. The resulting PCR fragmentwas digested with restriction enzymes BglII and MI, and the PCR fragmentwas ligated to the above-mentioned polylinker, to produce a DNA fragmentof about 400 bp. The ligation reaction was performed with a DNA LigationKit Ver.2.1 (manufactured by Takara Bio Inc.) under reaction conditionsrecommended by the manufacturer. Then, a fragment of about 9.2 kbp,obtained by cleaving pAYC32 (J. Gen. Microbiol., 137, 169-178 (1991))with restriction enzymes EcoRI and BamHI, was collected, and theabove-mentioned DNA fragment was inserted to construct the pAYCTER3expression plasmid, which is capable of functioning in M. methylotrophusNCIMB 10515. pAYCTER3 lacks a sequence upstream on the 5′ side of thestrA gene, and alternatively has a pUC19 multi-cloning site and an rrnBterminator.

Construction of pFLP31

The gentamicin resistance gene from the #403-11Gm strain constructed inReference Example 7 was designed so that it is located between two FRTsequences, so the drug resistance gene can be eliminated from thechromosome by a reaction with FLP recombinase. pAYCTER3 constructed bythe above-mentioned method was digested with BamHI-SmaI, and a 3.3-kbpfragment obtained by cleaving pCP20 (Proceedings of the National Academyof Sciences of the United States of America, (2000) 97, 6640-6645) withSmaI-BamHI containing an FLP recombinase was inserted. The resultingplasmid was designated as pFLP31. The plasmid pCP20 was registered atthe E. coli Genetic Stock Center as CGSC strain #7629, and it isavailable from the Center.

Elimination of the Antibiotic-Resistance Marker

pAET7 was eliminated from #403-11Gm by a known method to obtain astreptomycin-sensitive strain. The above-mentioned plasmid pFLP31 wasintroduced into the strain by electroporation, and the cells wereinoculated onto an SEII agar medium containing 50 mg/L streptomycin and0.5 g/L L-methionine. The resulting strain was suspended to anappropriate concentration in an SEII agar medium containing 50 mg/Lstreptomycin and 0.5 g/L L-methionine. The suspension was heated to 42°C. for 1 hour and diluted to form single colonies, and the cells wereinoculated onto an SEII agar medium containing 50 mg/L streptomycin and0.5 g/L L-methionine. From the colonies, strains sensitive to gentamicinwere selected. Then, pAET7 was eliminated from the strain to obtain astreptomycin-sensitive strain, which was designated as #403-11. When the#403-11 strain and a control strain (#403 strain) were cultured in anSEII production medium containing 0.075 g/L L-methionine and 2.5 g/Lsodium pyruvate, the amount of Lys that was produced increased. Theamount of Lys produced by the #403 strain was defined as 100, and therelative value of the amount of Lys produced by the #403-11 strain wascalculated and shown in Table 3.

TABLE 3 Relative amount of Strain produced Lys (%) #403 100 #403-11 108

Reference Example 10 Confirmation of Increased DDPS Activity

In #403-11 obtained as described above, 8 copies of the lysE24dapAcassette were transferred to the chromosome. Therefore, the activity ofdihydrodipicolinate synthase encoded by dapA was increased in the#403-11 strain. The dihydrodipicolinate synthase activity was measuredby modifying a known method (Journal of Biological Chemistry, 240,4710-4716 (1965)). Specifically, a reaction solution was prepared so asto contain 50 mM imidazole-HCl (pH 7.4), 2 mML-aspartate-β-semialdehyde, 2 mM sodium pyruvate, and a cell extract,and the final volume of the solution was adjusted to 1 ml. The resultsare shown in Table 4.

TABLE 4 Bacterial Specific activity strain (milliunit/milligram protein)AS1 12 #403-11 129

The amount of enzyme that produces 1 micromol of a product per minutewas defined as 1 unit.

Reference Example 11 Construction of the pBGEA Plasmid, and Constructionof #403-11/pBGEA

(1) Construction of the pBGEA Plasmid Carrying an L-Lysine BiosyntheticEnzyme Gene (dapA*) and a Gene Having L-Lysine Export Activity (lysE24)

In order to introduce the dapA* and LysE24 genes into a bacteriumbelonging to the genus Methylophilus, pBHR1 (Antoine, R. and Locht, C.,Molecular Microbiology, 6, 1785-99. (1992)) was used to construct pBGEAfor expressing dapA* and LysE24. First, pBHR1 was digested with the DraIrestriction enzyme, and the resulting fragment was added to aphenol/chloroform solution and mixed to terminate the reaction. Afterthe reaction mixture was centrifuged, the upper layer was collected, andDNA was collected by ethanol precipitation. The resulting DNA fragmentwas blunt-ended using a DNA Blunting kit (manufactured by Takara Shuzo).

The dapA* and LysE24 genes were obtained from pRSlysEdapA (JP 2003-61687A, U.S. Pat. No. 7,169,587). The E. coli JM109 strain transformed withthe pRSlysEdapA plasmid was designated as AJ13832 and deposited atNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary on Jun. 4, 2001 under theaccession number FERM P-18371. Then, the deposit was converted to aninternational deposit under the provisions of the Budapest Treaty on May13, 2002 under the accession number FERM BP-8042. First, pRSlysEdapA wasdigested with restriction enzymes EcoRI and BglII, and the resultingfragment was added to a phenol/chloroform solution and mixed toterminate the reaction. After the reaction mixture was centrifuged, theupper layer was collected, and DNA was collected by ethanolprecipitation. Then, the DNA fragment of interest was separated byelectrophoresis on an 0.8% agarose gel, and the DNA fragment of about2.0 Kbp was collected by using EASY TRAP ver. 2 (DNA collection kit,manufactured by Takara Shuzo). The resulting DNA fragment wasblunt-ended with a BKL kit (manufactured by Takara Shuzo) andphosphorylated.

The digestion product of pBHR1, and the dapA* and LysE24 gene regionfragments prepared as described above were ligated by using a DNALigation Kit Ver. 2 (manufactured by Takara Shuzo). This ligationproduct was used to transform Escherichia coli (E. coli JM109 competentcells, manufactured by Takara Shuzo), and the cells were inoculated intoan LB agar medium containing 20 mg/L of kanamycin and incubatedovernight at 37° C. The colonies that appeared on the agar medium wereinoculated into an LB liquid medium containing 20 mg/L of kanamycin andcultured at 37° C. for 8 hours with shaking. Plasmid DNA was extractedfrom each culture medium by the alkali-SDS method, and the structure ofeach plasmid was confirmed by digestion with restriction enzymes anddetermination of the nucleotide sequence. A plasmid with identicaltranscription directions of the chloramphenicol resistance gene and thedapA*and lysE24 genes was selected as pBHR-EA.

A gentamicin resistant marker was introduced into pBHR-EA obtained asdescribed above to construct the pBGEA plasmid. First, pBHR-EA wasdigested with the restriction enzyme NcoI, and the resulting fragmentwas added to a phenol/chloroform solution and mixed to terminate thereaction. After the reaction mixture was centrifuged, the upper layerwas collected, and DNA was collected by ethanol precipitation. Theresulting DNA fragment was blunt-ended by using a DNA Blunting kit(manufactured by Takara Shuzo). The gentamicin resistance gene regionwas amplified by PCR using pML122 (Monika Labes, Alfred Puhler, andReinhard Simon, Gene, 89, (1990), 37-46) as the template DNA and pGm-f(SEQ ID NO: 17) and pGm-r (SEQ ID NO: 18) as primers, and PCR wasperformed under the following conditions: denaturation at 94° C. for 10seconds, annealing at 60° C. for 30 seconds, and extension at 72° C. for90 seconds. The PCR was performed using Pyrobest DNA polymerase(manufactured by Takara Shuzo). The resulting gentamicin resistance genefragment was blunt-ended by using a BKL kit (manufactured by TakaraShuzo) and phosphorylated.

The digestion product of pBHR-EA and the gentamicin resistance generegion fragment prepared as described above were ligated by using a DNALigation Kit Ver. 2 (manufactured by Takara Shuzo). This ligationproduct was used to transform Escherichia coli (E. coli JM109 competentcells, manufactured by Takara Shuzo), and the cells were inoculated ontoan LB agar medium containing 20 mg/L of gentamicin and incubatedovernight at 37° C. The colonies that appeared on the agar medium wereinoculated into an LB liquid medium containing 20 mg/L of gentamicin andcultured at 37° C. for 8 hours with shaking. Plasmid DNA was extractedfrom each culture medium by the alkali-SDS method, and the structure ofeach plasmid was confirmed by digestion with restriction enzymes anddetermination of the nucleotide sequence to obtain pBGEA. pBGEA was thenintroduced into the #403-11 strain prepared in Example 9, in which thelysE24 and dapA* genes had been incorporated into the chromosome, toenhance the lysE24 and dapA* genes. The strain was designated as#403-11/pBGEA. When #403-11/pBGEA and the control strain #403-11 werecultured in an SEII production medium containing 20 mg/L kanamycin, 50mg/L gentamicin (the medium for the control strain contains nogentamicin), 0.075 g/L L-methionine, and 2.5 g/L sodium pyruvate, theamount of Lys increased. The amount of Lys produced by the #403-11strain was defined as 100, and the relative value of the amount of Lysproduced by the #403-11/pBGEA strain was calculated and shown in Table5. It was found that the introduction of the plasmid increased theamount of Lys produced by the #403-11 strain.

TABLE 5 Relative amount of Strain produced Lys (%) #403-11 100#403-11/pBGEA 111

Reference Example 12 Construction of the pRSlysA, pRSddh, pRSdapB,pRSasd, and pRSask Plasmids, Introduction of the Plasmids into the#403-11/pBGEA Strain, and Evaluation of L-Lysine Productivity

Next, expression plasmids carrying each Lys biosynthetic gene wereconstructed, and the plasmids were introduced into the #403-11/pBGEAstrain, and the effects on L-lysine productivity were investigated.

<1> Construction of the pRSlysA Plasmid

The diaminopimelate decarboxylase gene (lysA) from Methylophilusmethylotrophus was obtained by PCR using two oligonucleotide primersprepared based on a known sequence (SEQ ID NO: 13 in WO2000/061723) andusing the chromosomal DNA of Methylophilus methylotrophus as thetemplate. PCR was performed using plysA-f (SEQ ID NO: 19:) and plysA-r(SEQ ID NO: 20) as primers, under the following conditions for 25cycles: denaturation at 94° C. for 20 seconds, annealing at 55° C. for30 seconds, and extension at 72° C. for 60 seconds. The amplified lysAgene fragment from M. methylotrophus was digested with Sse8387I-XbaI,recognition sites of which had been added into the primer sequence, andthe resulting fragment was ligated to pRStac (JP 2003-61687 A, U.S. Pat.No. 7,169,587) which had been digested with Sse8387I-XbaI. The plasmidwas designated as pRSlysA.

<2> pRSddh

The diaminopimelate hydrogenase gene (ddh) from Brevibacteriumlactofermentum was obtained by amplification with PCR using two kinds ofoligonucleotide primers prepared based on the known nucleotide sequenceof ddh based on Corynebacterium glutamicum (Ishino, S. et al. Nucleicacid res. 15, 3917 (1987)) and using the chromosomal DNA ofBrevibacterium lactofermentum 2256 strain (ATCC 13869 strain) as thetemplate. PCR was performed using pddh-f (SEQ ID NO: 21) and pddh-r (SEQID NO: 22) as primers, under the following conditions for 25 cycles:denaturation at 94° C. for 20 seconds, annealing at 55° C. for 30seconds, and extension at 72° C. for 60 seconds. The amplified ddh genefragment was digested with Sse83871-XbaI, recognition sites of which hadbeen added into the primer sequence, and the resulting fragment wasligated to pRStac (JP 2003-61687 A, U.S. Pat. No. 7,169,587) digestedwith Sse83871-XbaI. The plasmid was designated as pRSddh.

<3> pRSdapB

The dihydrodipicolinate reductase gene (dapB) from E. coli was amplifiedby PCR using two oligonucleotide primers prepared based on the knownnucleotide sequence and using the chromosomal DNA of E. coli as thetemplate. PCR was performed using pdapB-f (SEQ ID NO: 23:) and pdapB-r(SEQ ID NO: 24) as primers, under the following conditions for 25cycles: denaturation at 94° C. for 20 seconds, annealing at 55° C. for30 seconds, and extension at 72° C. for 60 seconds. The amplified dapBgene fragment was digested with Sse83871-XbaI, recognition sites ofwhich had been added into the primer sequence, and the resultingfragment was ligated to pRStac (JP 2003-61687 A, U.S. Pat. No.7,169,587) which had been digested with Sse83871-XbaI. The plasmid wasdesignated as pRSdapB.

<4> pRSlasd

The aspartate-semialdehyde dehydrogenase gene (asd) from E. coli wasamplified by PCR using two oligonucleotide primers prepared based on theknown nucleotide sequence using the chromosomal DNA of E. coli as thetemplate. PCR was performed using pasd-f (SEQ ID NO: 25) and pasd-r (SEQID NO: 26) as primers, under the following conditions for 25 cycles:denaturation at 94° C. for 20 seconds, annealing at 55° C. for 30seconds, and extension at 72° C. for 60 seconds. The amplified ddh genefragment was digested with Sse83871-XbaI, reconition sites of which hadbeen added into the primer sequence, and the resulting fragment wasligated to pRStac (JP 2003-61687 A, U.S. Pat. No. 7,169,587) which hadbeen digested with Sse83871-XbaI. The plasmid was designated as pRSasd.

<5> pRS ask

The aspartokinase gene (ask) from Methylophilus methylotrophus wasobtained by PCR using two oligonucleotide primers prepared based on aknown sequence (SEQ ID NO: 5 in WO2000/061723) and using a chromosomalDNA of Methylophilus methylotrophus as a template. PCR was performedusing pask-f (SEQ ID NO: 27:) and pask-r (SEQ ID NO: 28:) as primers.The following cycle was repeated 25 times: denaturation at 94° C. for 20seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for90 seconds. The amplified ask gene fragment from M. methylotrophus wasdigested with Sse8387I-XbaI, recognition sites of which had been addedinto the primer and blunt-ended, and the resulting fragment was ligatedto pRStac (JP 2003-61687 A, U.S. Pat. No. 7,169,587) which had beendigested with Sse8387I and blunt-ended. The plasmid was designated aspRS ask.

<6> Introduction and Evaluation of the Plasmids

The five plasmids prepared as described above were separately introducedinto the #403-11/pBGEA strain prepared in Example 11, and the resultingstrains were cultured in an SEII production medium containing 0.075 g/LL-methionine and 2.5 g/L sodium pyruvate. However, Lys productivity didnot improve in all the strains. In the strain with ddh, Lys productivitywas reduced.

Reference Example 13 Enhancement of ddh+lysA in Combination,Construction of the pDA Plasmid, and Evaluation of Productivity ofL-Lysine

The #403-11 strain was modified so that expression of the lysE24 anddapA* genes, which are effective for improving Lys production in M.methylotrophus, was sufficiently enhanced by gene incorporation andplasmid introduction. This strain was used to determine the nextlimiting factor in Lys production. As shown in Reference Example 12,when the genes were separately increased, the effective gene could notbe determined. Therefore, various plasmids were constructed to enhancethe genes in combination, each carrying two genes, and introduced intothe #403-11 strain. As a result, it was found that by increasing theexpression of both the lysA and ddh genes, the productivity of L-lysinewas improved.

Diaminopimelate dehydrogenase encoded by ddh was thought to reversiblycatalyze both the production and degradation reactions of diaminopimelicacid, and that enhancing ddh alone promoted not only production but alsodegradation of diaminopimelic acid. On the other hand, diaminopimelatedecarboxylase, which catalyzes the reaction subsequent to that ofdiaminopimelate dehydrogenase, is an irreversible enzyme that causes adecarboxylation reaction prior to L-lysine production. It was thoughtthat enhancing both diaminopimelate dehydrogenase and diaminopimelatedecarboxylase together prevented degradation of diaminopimelic acid bydiaminopimelate dehydrogenase and promoted synthesis of diaminopimelicacid.

Specifically, the pRSddh plasmid was digested with SapI, and theresulting fragment was blunt-ended and dephosphorylated to produce avector, into which a PCR-amplified DNA fragment including the lysA generegion containing the tac promoter region. PCR amplification wasperformed using the pRSlysA plasmid as the template and ptac-f (SEQ IDNO: 29:) and plysA-r (SEQ ID NO: 20:) as primers. The following cyclewas repeated 25 times: denaturation at 94° C. for 20 seconds, annealingat 55° C. for 30 seconds, and extension at 72° C. for 60 seconds. Theresulting PCR-amplified fragment was digested with XbaI, and theresulting fragment was blunt-ended and ligated to the above-mentionedvector. In the resulting plasmid, the transcription directions of theddh and lysA genes were identical, and the plasmid was designated aspDA. When a strain into which the pDA was introduced,(#403-11/pBGEA/pDA) and the control strain #403-11/pBGEA, were culturedin an SEII production medium containing 20 mg/L kanamycin, 50 mg/Lgentamicin, and 50 mg/L streptomycin (the medium for the control straincontains no streptomycin), 0.075 g/L L-methionine, and 2.5 g/L sodiumpyruvate, the amount of Lys increased. The amount of Lys produced by the#403-11/pBGEA strain was defined as 100, and the relative value of theamount of Lys produced by the #403-11/pBGEA/pDA strain was calculatedand shown in Table 6.

TABLE 6 Relative amount of Strain produced Lys (%) #403-11/pBGEA 100#403-11/pBGEA/pDA 120

Reference Example 14 Enhancement of Further L-Lysine Biosynthetic Genesin Combination, Construction of pBDAS (lysA+ddh+dapB+asd), andEvaluation of L-Lysine Productivity

It was found that the L-lysine productivity was improved by increasingthe expression of both the ddh and lysA genes in combination, sincethese genes are capable of catalyzing two sequential reactions in thebiosynthesis pathway. The effect of enhancing other enzyme genes incombination with ddh+lysA was investigated and it was found that the useof dapB and asd in combination with ddh and lysA improved L-lysineproduction.

Specifically, the pRSdapB plasmid was digested with EcoRI, and theresulting fragment was blunt-ended and dephosphorylated to prepare avector. The plasmid pDA was digested with HpaI-SapI, and the resultingDNA fragment of 2.5 kbp, which includes lysA and ddh which each have atac promoter upstream of the genes, was collected and blunt-ended to beligated to the vector, to thereby construct pBDA. The plasmid was foundto have the ddh and lysA genes inserted upstream of dapB so that thedirection of ddh and lysA and the direction of dapB were identical. ThepBDA plasmid was further digested with SapI, and the resulting fragmentwas blunt-ended and dephosphorylated to prepare a vector. APCR-amplified DNA fragment including the asd gene region containing atac promoter region was then inserted into the vector. PCR amplificationwas performed using the pRSasd plasmid as the template and ptac-f andpasd-r as primers. The following cycle was repeated 25 times:denaturation at 94° C. for 20 seconds, annealing at 55° C. for 30seconds, and extension at 72° C. for 90 seconds. The resultingPCR-amplified fragment was blunt-ended and phosphorylated, and thefragment was ligated to the above-mentioned vector. The resultingplasmid was designated as pBDAS. When a strain into which the pBDASplasmid was introduced (#403-11/pBGEA/pBDAS), and a control strain#403-11/pBGEA, were cultured in an SEII production medium containing 20mg/L kanamycin, 50 mg/L gentamicin, and 50 mg/L streptomycin (the mediumfor the control strain contains no streptomycin), 0.075 g/LL-methionine, and 2.5 g/L sodium pyruvate, the amount of Lys increased.The amount of Lys produced by the #403-11/pBGEA strain was defined as100, and the relative value of the amount of Lys produced by the#403-11/pBGEA/pBDAS strain was calculated and is shown in Table 7.

TABLE 7 Relative amount of Strain produced Lys (%) #403-11/pBGEA 100#403-11/pBGEA/pDA 120 #403-11/pBGEA/pBDAS 143

Example: 1 The Effect on Fermentation Results of the Rate of Increase inIonic Concentration

The effect on L-lysine fermentation of the rate of increase in ionicconcentration was determined with the #403-11/pBGEA/pBDAS strain.

First, all of the bacterial cells obtained by culturing the#403-11/pBGEA/pBDAS strain in SEII agar medium for 24 hours at 37° C.were scraped off a single plate, and the entire suspension solution wasinoculated into a 1-liter jar fermenter containing 300 mL of mainculture medium, and culturing was conducted at 34° C. at pH 6.6. Thecomposition of the main culture medium is indicated below.

Main Culture Medium:

K₂HPO₄ 1.9 g/L NaH₂PO₄ 1.56 g/L Sodium pyruvate 2.5 g/L MgSO₄•7H₂O 0.2g/L (NH₄)₂SO₄ 4.97 g/L L-methionine 0.65 g/L Citric acid 1 g/LMgSO₄•7H₂O [sic] 1 g/L CuSO₄•5H₂O 25 μg/L MnSO₄•5H₂O 125 μg/L ZnSO₄•7H₂O115 μg/L CaCl₂•2H₂O 0.36 g/L FeCl₃•6H₂O 48.5 mg/L Methanol 2% (vol/vol)Streptomycin sulfate 50 mg/L Gentamicin sulfate 50 mg/L Kanamycinsulfate 25 mg/L

Components other than methanol and the antibiotics were mixed,sterilized by filtration. Subsequently, methanol and filter-sterilizedantibiotics were added.

During culturing, the pH was adjusted to 6.6 by the addition of ammoniagas. When the methanol in the medium dropped to 0.2% or below, a 100percent methanol solution was fed by fed-batch culturing.

In Lys fermentation, a nitrogen source was added to the medium for Lysproduction and ammonium sulfate was added to the medium to supplycounter ions for the targeted amino acid. In the course of addingammonium sulfate to the medium, the effect of feeding resulted in therate of increase in total ionic strength in the medium of 0.03mol/m³/hour, 0.02 mol/m³/hour, and 0.012 mol/m³/hour were examined.

The results are given in Table 8. The production is given as a relativevalue where production under conditions of a rate of increase in ionicstrength of 0.03 mol/m³/hour was adopted as 100 percent. As shown inTable 1, as the rate of increase in ionic strength dropped, productionwas found to rise. As a result, it was determined that L-lysineproduction was enhanced by conducting feeding so that the rate ofincrease in ionic strength in the medium was maintained at 0.02mol/m³/hour, desirably 0.012 mol/m³/hour or less.

TABLE 8 Rate of increase in ionic Production Relative strength(mol/m³/hour) value (%) 0.03 100 0.02 106 0.012 133

Example: 2

To examine the effect of the reduced rate of increase in ionic strengthon the fermentation, a nitrogen source of ammonium chloride and glutamicacid was employed as a monovalent counter ion. The glutamic acid wasemployed in the form of a solution that had been neutralized withammonia.

First, in the same manner as in Example 1, All of the bacterial cellsobtained by culturing the #403-11/pBGEA/pBDAS strain in SEII agar mediumfor 24 hours at 37° C. were scraped up, and the entire suspensionsolution was inoculated into a 1-liter jar fermenter containing 300 mLof main culture to 6.6 medium, and culturing was conducted at pH 6.6 and34° C. The pH was adjusted during culturing by the addition of ammoniagas. When the methanol in the medium dropped to 0.2% or below, a 100%methanol solution was fed to conduct fed-batch culturing.

In the course of adding nitrogen sources in the form of ammoniumchloride and glutamic acid to the medium, these compounds were fed so asto achieve rates of increase in ionic strength of 0.008 mol/m³/hour and0.006 mol/m³/hour.

The results are given in Table 9. The production is given as a relativevalue where production under conditions of a rate of increase in ionicstrength of 0.03 mol/m³/hour with the use of ammonium sulfate wasadopted as 100 percent. As a result of the test, it was confirmed thatan increase in the rate of ionic strength of 0.012 mol/m³/hour or lessfurther increased production.

TABLE 9 Rate of increase in ionic Production Relative Nitrogen sourcestrength (mol/m³/hour) value (%) Ammonium sulfate 0.03 100 Ammoniumsulfate 0.02 106 Ammonium sulfate 0.012 133 Ammonium chloride 0.008 148Glutamic acid 0.006 147

INDUSTRIAL APPLICABILITY

The present invention enhances the production of carboxylic acid bymethanol-assimilating bacteria.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, an equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments is incorporated by reference herein in its entirety.

We claim:
 1. A method for producing carboxylic acid by fermentationcomprising, A) culturing a methanol-assimilating bacterium that has anability to produce the carboxylic acid in a liquid medium containingmethanol and a counter ion, B) collecting the carboxylic acid from themedium or the bacterium, wherein a composition comprising methanol and acounter ion is fed to the medium by fed-batch culturing.
 2. The methodof claim 1, wherein said counter ion is a monovalent ion.
 3. The methodaccording to claim 1, wherein said counter ion is selected from thegroup consisting of ammonium chloride, ammonium glutamate, ammoniumsuccinate, ammonium fumarate, ammonium aspartate, and combinationsthereof.
 4. The method according to claim 1, wherein said carboxylicacid is an L-amino acid.
 5. The method according to claim 1, whereinsaid L-amino acid is L-lysine.
 6. The method according to claim 1,wherein said methanol-assimilating bacterium is of a genera selectedfrom the group consisting Acromobacter, Pseudomonas, Protaminobacter,Methanomonas, Microcyclus, Methylobacillus, Bacillus, and Methylophilus.7. The method according to claim 1, wherein said methanol-assimilatingbacterium has been modified to increase the activity of an enzymeselected from the group consisting of diaminopimelate dehydrogenase,diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, andcombinations thereof.
 8. The method according to claim 6, wherein DNAencoding dihydrodipicolinate synthase and/or aspartokinase that has beenmodified so it is not subject to feedback inhibition by L-lysine, ispresent in the bacterium.
 9. The method according to claim 7, whereinDNA encoding a variant lysE protein promoting the export of L-lysine tothe outside of the cell when introduced into a methanol-assimilatingbacterium, is present in the bacterium.